IMPROVED ADENO-ASSOCIATED VIRUS-BINDING PROTEIN, METHOD FOR PRODUCING SAME, AND ADENO-ASSOCIATED VIRUS ADSORBENT USING SAME

Abstract

The present invention provides an improved adeno-associated virus (AAV)-binding protein having enhanced stability, especially, heat stability, acid stability and alkali stability, of an AAV-binding protein, a method for producing the improved AAV-binding protein, and an AAV adsorbent using the same.

Claims

1. An adeno-associated virus (AAV)-binding protein which is selected from any one of the following (i) to (iii): (i) an AAV-binding protein comprising at least amino acid residues from serine at position 312 to aspartic acid at position 500 of an amino acid sequence set forth in SEQ ID NO: 1, wherein at least any one of the following amino acid substitutions (1) to (137) is present at the amino acid residues from positions 312 to 500: (1) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, (2) a substitution of isoleucine with phenylalanine at position 319 of SEQ ID NO: 1, (3) a substitution of leucine with proline at position 321 of SEQ ID NO: 1, (4) a substitution of proline with leucine at position 322 of SEQ ID NO: 1, (5) a substitution of asparagine with aspartic acid or serine at position 324 of SEQ ID NO: 1, (6) a substitution of glutamine with arginine at position 327 of SEQ ID NO: 1, (7) a substitution of asparagine with aspartic acid or tyrosine at position 329 of SEQ ID NO: 1, (8) a substitution of alanine with glycine at position 330 of SEQ ID NO: 1, (9) a substitution of tyrosine with cysteine at position 331 of SEQ ID NO: 1, (10) a substitution of valine with glutamine at position 332 of SEQ ID NO: 1, (11) a substitution of leucine with valine or proline at position 333 of SEQ ID NO: 1, (12) a substitution of glutamine with arginine at position 334 of SEQ ID NO: 1, (13) a substitution of proline with leucine at position 337 of SEQ ID NO: 1, (14) a substitution of lysine with arginine or glutamic acid at position 338 of SEQ ID NO: 1, (15) a substitution of glutamic acid with glycine at position 340 of SEQ ID NO: 1, (16) a substitution of threonine with alanine at position 341 of SEQ ID NO: 1, (17) a substitution of tyrosine with cysteine, histidine, or asparagine at position 342 of SEQ ID NO: 1, (18) a substitution of threonine with methionine at position 343 of SEQ ID NO: 1, (19) a substitution of tyrosine with histidine at position 344 of SEQ ID NO: 1, (20) a substitution of aspartic acid with asparagine at position 345 of SEQ ID NO: 1, (21) a substitution of tryptophan with leucine or arginine at position 346 of SEQ ID NO: 1, (22) a substitution of glutamine with leucine or proline at position 347 of SEQ ID NO: 1, (23) a substitution of leucine with proline at position 348 of SEQ ID NO: 1, (24) a substitution of isoleucine with threonine at position 349 of SEQ ID NO: 1, (25) a substitution of threonine with methionine at position 350 of SEQ ID NO: 1, (26) a substitution of histidine with leucine at position 351 of SEQ ID NO: 1, (27) a substitution of proline with leucine at position 352 of SEQ ID NO: 1, (28) a substitution of arginine with cysteine at position 353 of SEQ ID NO: 1, (29) a substitution of aspartic acid with glycine at position 354 of SEQ ID NO: 1, (30) a substitution of tyrosine with histidine, asparagine, or cysteine at position 355 of SEQ ID NO: 1, (31) a substitution of serine with cysteine at position 356 of SEQ ID NO: 1, (32) a substitution of glycine with cysteine at position 357 of SEQ ID NO: 1, (33) a substitution of histidine with arginine or leucine at position 363 of SEQ ID NO: 1, (34) a substitution of serine with proline at position 364 of SEQ ID NO: 1, (35) a substitution of glutamine with arginine at position 365 of SEQ ID NO: 1, (36) a substitution of isoleucine with threonine at position 366 of SEQ ID NO: 1, (37) a substitution of leucine with proline at position 367 of SEQ ID NO: 1, (38) a substitution of lysine with arginine at position 368 of SEQ ID NO: 1, (39) a substitution of leucine with glutamine or proline at position 369 of SEQ ID NO: 1, (40) a substitution of serine with alanine at position 370 of SEQ ID NO: 1, (41) a substitution of lysine with glutamic acid at position 371 of SEQ ID NO: 1, (42) a substitution of threonine with alanine at position 373 of SEQ ID NO: 1, (43) a substitution of leucine with proline at position 376 of SEQ ID NO: 1, (44) a substitution of tyrosine with cysteine at position 377 of SEQ ID NO: 1, (45) a substitution of phenylalanine with serine at position 379 of SEQ ID NO: 1, (46) a substitution of valine with alanine at position 381 of SEQ ID NO: 1, (47) a substitution of glutamic acid with glycine at position 384 of SEQ ID NO: 1, (48) a substitution of asparagine with serine at position 387 of SEQ ID NO: 1, (49) a substitution of histidine with glutamine, leucine, or arginine at position 389 of SEQ ID NO: 1, (50) a substitution of glutamic acid with lysine at position 391 of SEQ ID NO: 1, (51) a substitution of tyrosine with cysteine at position 393 of SEQ ID NO: 1, (52) a substitution of valine with alanine at position 396 of SEQ ID NO: 1, (53) a substitution of lysine with glutamic acid or arginine at position 399 of SEQ ID NO: 1, (54) a substitution of arginine with serine at position 406 of SEQ ID NO: 1, (55) a substitution of valine with alanine at position 412 of SEQ ID NO: 1, (56) a substitution of glutamine with leucine at position 415 of SEQ ID NO: 1, (57) a substitution of phenylalanine with serine at position 416 of SEQ ID NO: 1, (58) a substitution of glutamine with leucine at position 441 of SEQ ID NO: 1, (59) a substitution of tyrosine with phenylalanine at position 442 of SEQ ID NO: 1, (60) a substitution of lysine with arginine at position 448 of SEQ ID NO: 1, (61) a substitution of glutamic acid with glycine at position 453 of SEQ ID NO: 1, (62) a substitution of lysine with arginine at position 455 of SEQ ID NO: 1, (63) a substitution of glutamic acid with glycine at position 458 of SEQ ID NO: 1, (64) a substitution of alanine with serine at position 461 of SEQ ID NO: 1, (65) a substitution of serine with arginine at position 476 of SEQ ID NO: 1, (66) a substitution of leucine with proline at position 477 of SEQ ID NO: 1, (67) a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (68) a substitution of asparagine with aspartic acid at position 492 of SEQ ID NO: 1, (69) a substitution of isoleucine with asparagine or serine at position 319 of SEQ ID NO: 1, (70) a substitution of threonine with isoleucine at position 320 of SEQ ID NO: 1, (71) a substitution of lysine with glutamic acid at position 323 of SEQ ID NO: 1, (72) a substitution of leucine with glutamine or proline at position 328 of SEQ ID NO: 1, (73) a substitution of tyrosine with histidine at position 331 of SEQ ID NO: 1, (74) a substitution of valine with alanine or glutamic acid at position 332 of SEQ ID NO: 1, (75) a substitution of proline with glutamine at position 336 of SEQ ID NO: 1, (76) a substitution of proline with glutamine at position 337 of SEQ ID NO: 1, (77) a substitution of lysine with asparagine at position 338 of SEQ ID NO: 1, (78) a substitution of tyrosine with arginine at position 342 of SEQ ID NO: 1, (79) a substitution of threonine with serine at position 350 of SEQ ID NO: 1, (80) a substitution of isoleucine with phenylalanine at position 366 of SEQ ID NO: 1, (81) a substitution of valine with alanine at position 383 of SEQ ID NO: 1, (82) a substitution of glutamine with arginine at position 386 of SEQ ID NO: 1, (83) a substitution of histidine with aspartic acid at position 389 of SEQ ID NO: 1, (84) a substitution of glycine with cysteine at position 392 of SEQ ID NO: 1, (85) a substitution of valine with alanine at position 394 of SEQ ID NO: 1, (86) a substitution of isoleucine with valine at position 409 of SEQ ID NO: 1, (87) a substitution of serine with glycine at position 476 of SEQ ID NO: 1, (88) a substitution of threonine with serine at position 490 of SEQ ID NO: 1, (89) a substitution of serine with proline at position 312 of SEQ ID NO: 1, (90) a substitution of alanine with serine at position 313 of SEQ ID NO: 1, (91) a substitution of valine with aspartic acid at position 317 of SEQ ID NO: 1, (92) a substitution of glutamine with proline at position 318 of SEQ ID NO: 1, (93) a substitution of threonine with alanine at position 320 of SEQ ID NO: 1, (94) a substitution of lysine with arginine at position 323 of SEQ ID NO: 1, (95) a substitution of valine with alanine or glutamic acid at position 326 of SEQ ID NO: 1, (96) a substitution of glutamine with histidine or leucine at position 327 of SEQ ID NO: 1, (97) a substitution of asparagine with histidine or isoleucine at position 329 of SEQ ID NO: 1, (98) a substitution of valine with alanine at position 332 of SEQ ID NO: 1, (99) a substitution of glutamic acid with glycine or valine at position 335 of SEQ ID NO: 1, (100) a substitution of glutamic acid with valine at position 340 of SEQ ID NO: 1, (101) a substitution of threonine with proline at position 341 of SEQ ID NO: 1, (102) a substitution of threonine with serine at position 343 of SEQ ID NO: 1, (103) a substitution of tyrosine with phenylalanine at position 344 of SEQ ID NO: 1, (104) a substitution of tryptophan with cysteine at position 346 of SEQ ID NO: 1, (105) a substitution of methionine with leucine at position 359 of SEQ ID NO: 1, (106) a substitution of glutamic acid with lysine or valine at position 360 of SEQ ID NO: 1, (107) a substitution of glycine with cysteine at position 361 of SEQ ID NO: 1, (108) a substitution of lysine with glutamic acid, asparagine, or glycine at position 362 of SEQ ID NO: 1, (109) a substitution of serine with leucine at position 364 of SEQ ID NO: 1, (110) a substitution of lysine with asparagine or aspartic acid at position 371 of SEQ ID NO: 1, (111) a substitution of leucine with proline or glutamine at position 372 of SEQ ID NO: 1, (112) a substitution of proline with leucine at position 374 of SEQ ID NO: 1, (113) a substitution of glutamic acid with glycine or valine at position 378 of SEQ ID NO: 1, (114) a substitution of phenylalanine with tyrosine or cysteine at position 379 of SEQ ID NO: 1, (115) a substitution of lysine with glutamic acid at position 380 of SEQ ID NO: 1, (116) a substitution of valine with aspartic acid at position 381 of SEQ ID NO: 1, (117) a substitution of isoleucine with valine at position 382 of SEQ ID NO: 1, (118) a substitution of glutamic acid with valine at position 384 of SEQ ID NO: 1, (119) a substitution of valine with aspartic acid or isoleucine at position 394 of SEQ ID NO: 1, (120) a substitution of asparagine with serine at position 395 of SEQ ID NO: 1, (121) a substitution of threonine with serine at position 397 of SEQ ID NO: 1, (122) a substitution of glutamic acid with valine at position 401 of SEQ ID NO: 1, (123) a substitution of arginine with histidine at position 403 of SEQ ID NO: 1, (124) a substitution of arginine with histidine at position 406 of SEQ ID NO: 1, (125) a substitution of glutamine with arginine at position 415 of SEQ ID NO: 1, (126) a substitution of threonine with alanine at position 426 of SEQ ID NO: 1, (127) a substitution of glutamine with leucine or arginine at position 432 of SEQ ID NO: 1, (128) a substitution of glutamine with arginine at position 441 of SEQ ID NO: 1, (129) a substitution of histidine with leucine at position 443 of SEQ ID NO: 1, (130) a substitution of lysine with glutamic acid at position 448 of SEQ ID NO: 1, (131) a substitution of isoleucine with valine at position 456 of SEQ ID NO: 1, (132) a substitution of aspartic acid with asparagine at position 483 of SEQ ID NO: 1, (133) a substitution of serine with leucine at position 488 of SEQ ID NO: 1, (134) a substitution of valine with glutamic acid or isoleucine at position 499 of SEQ ID NO: 1; (ii) an AAV-binding protein comprising at least amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein at least any one of the amino acid substitutions (1) to (134) is present at the amino acid residues from positions 312 to 500, wherein one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues are further present at one or several positions other than the amino acid substitutions shown in (1) to (134), and wherein the AAV-binding protein has AAV-binding activity; and (iii) an AAV-binding protein comprising an amino acid sequence, wherein the amino acid sequence has 70% or more homology to an entire amino acid sequence in which at least any one of the amino acid substitutions (1) to (134) is present in an amino acid sequence ranging from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the at least any one of the amino acid substitutions remains in the amino acid sequence, and wherein the AAV-binding protein has AAV-binding activity.

2. The AAV-binding protein according to claim 1, which is selected from any one of the following (iv) to (vi): (iv) an AAV-binding protein comprising at least amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein at least the following amino acid substitution (1) is present at the amino acid residues from positions 312 to 500: (1) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, (v) an AAV-binding protein comprising at least amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein at least the amino acid substitution (1) is present at the amino acid residues from positions 312 to 500, wherein one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues are further present at one or several positions other than the amino acid substitution shown in (1), and wherein the AAV-binding protein has AAV-binding activity; and (vi) an AAV-binding protein comprising an amino acid sequence, wherein the amino acid sequence has 70% or more homology to an entire amino acid sequence in which at least the amino acid substitution (1) is present in an amino acid sequence ranging from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid substitution (1) remains in the amino acid sequence, and wherein the AAV-binding protein has AAV-binding activity.

3. The AAV-binding protein according to claim 2, wherein at least any one of amino acid substitutions shown in the following (A) to (K) is present: (A) a substitution of glycine with serine at position 390 of SEQ ID NO: 1 and a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, (B) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, and a substitution of serine with arginine at position 476 of SEQ ID NO: 1, (C) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of isoleucine with phenylalanine at position 319 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, and a substitution of serine with arginine at position 476 of SEQ ID NO: 1, (D) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (E) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of isoleucine with phenylalanine at position 319 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 323 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, and a substitution of serine with arginine at position 476 of SEQ ID NO: 1, (F) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (G) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of asparagine with aspartic acid at position 324 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 362 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (H) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of valine with aspartic acid at position 317 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of lysine with asparagine at position 371 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (I) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of valine with aspartic acid at position 317 of SEQ ID NO: 1, a substitution of asparagine with aspartic acid at position 324 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 362 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, (J) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of valine with aspartic acid at position 317 of SEQ ID NO: 1, a substitution of tyrosine with cysteine at position 342 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 362 of SEQ ID NO: 1, a substitution of lysine with asparagine at position 371 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1, and (K) a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of valine with aspartic acid at position 317 of SEQ ID NO: 1, a substitution of tyrosine with serine at position 342 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 362 of SEQ ID NO: 1, a substitution of lysine with asparagine at position 371 of SEQ ID NO: 1, a substitution of lysine with alanine at position 381 of SEQ ID NO: 1, a substitution of isoleucine with valine at position 382 of SEQ ID NO: 1, a substitution of glycine with serine at position 390 of SEQ ID NO: 1, a substitution of lysine with glutamic acid at position 399 of SEQ ID NO: 1, a substitution of serine with arginine at position 476 of SEQ ID NO: 1, and a substitution of asparagine with aspartic acid at position 487 of SEQ ID NO: 1.

4. The AAV-binding protein according to claim 3, which is selected from any one of the following (vii) to (ix): (vii) an AAV-binding protein comprising at least amino acid residues from serine at position 25 to aspartic acid at position 213 of an amino acid sequence set forth in any one of SEQ ID NOS: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76; (viii) an AAV-binding protein comprising at least amino acid residues from serine at position 25 to aspartic acid at position 213 of an amino acid sequence set forth in any one of SEQ ID NOS: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, wherein one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues are further present at one or several positions of the amino acid residues from positions 25 to 213 other than the amino acid substitution in the amino acid sequence, and wherein the AAV-binding protein has AAV-binding activity; and (ix) an AAV-binding protein comprising amino acid residues having 70% or more homology to at least amino acid residues from serine at position 25 to aspartic acid at position 213 of an amino acid sequence set forth in any one of SEQ ID NOS: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, wherein one or more amino acid substitutions of said amino acid sequence are present, and having AAV-binding activity.

5. A polynucleotide encoding the AAV-binding protein according to claim 1.

6. An expression vector comprising the polynucleotide according to claim 5.

7. A transformant obtained by transforming a host with the expression vector according to claim 6.

8. The transformant according to claim 7, wherein the host is Escherichia coli.

9. A method for producing an AAV-binding protein, comprising a step of allowing the AAV-binding protein to be expressed by culturing the transformant according to claim 7 and a step of recovering the AAV-binding protein expressed from the obtained culture.

10. An AAV adsorbent comprising an insoluble carrier and the AAV-binding protein according to claim 1 which is immobilized on the carrier.

11. A column containing the AAV adsorbent according to claim 10.

12. A method for purifying AAV, comprising a step of adding a solution containing AAV to the column according to claim 11 to allow the AAV to be adsorbed by the adsorbent and a step of eluting the AAV adsorbed by the adsorbent using an eluate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0081] FIG. 1 is a graph showing the results of evaluating the binding activity of an adeno-associated virus (AAV)-binding protein to VLP2 (virus-like particles of AAV2) by an ELISA method. In the figure, (+) refers to the result when VLP2 is immobilized on the solid phase, and (−) refers to the result when VLP2 is not immobilized on the solid phase.

[0082] FIG. 2 is a graph showing a chromatographic pattern of AAV2 contained in a cell disruption solution purified by an AVR5e column. The peak near an elution time of 20 minutes was the AAV2 elution peak, and the fractions (Fr6-Fr9; 1 mL each) corresponding to the peak was recovered.

[0083] FIG. 3 is a profile showing the results of confirming the purification purity of AAV2 contained in the fractions (Fr6-Fr9) recovered in FIG. 2 by SDS-PAGE. AP in the figure is the analysis result of a solution obtained by diluting the sample before purification 100 times, and Fr6 to Fr9 are the analysis results of the respective fractions obtained in FIG. 2. VP1 to VP3 indicate the positions of the bands corresponding to the three types of capsid proteins constituting AAV.

[0084] FIG. 4 is a graph showing the results of recovering AAV2 contained in the solution using a column packed with a wild-type AAV-binding protein-immobilized gel or AVR5eHC-immobilized gel.

[0085] FIG. 5 is a graph showing the results of recovering AAV2 contained in the solution using a column packed with a wild-type AAV-binding protein-immobilized gel, AVR3HC-immobilized gel, or AVR8gHC-immobilized gel.

EXAMPLES

[0086] Hereinafter, examples will be shown to explain the invention in more detail, but the invention is not limited to these examples.

Example 1 Preparation of Adeno-Associated Virus (AAV)-Binding Protein Expression Vector

[0087] (1) The human codons of the nucleotide sequence encoding the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 corresponding to the extracellular domains 1 and 2 (PKD1 and PKD2) in the amino acid sequence of the AAV-binding protein set forth in SEQ ID NO: 1 (UniProt Accession No. Q8IZA0) were substituted with the Escherichia coli (E. coli) codons thereof. The codon-substituted nucleotide sequence is set forth in SEQ ID NO: 2.

[0088] (2) The gene set forth in SEQ ID NO: 2 was artificially totally synthesized and cloned into a plasmid (consigned to Eurofins Genomics K.K.). The prepared plasmid was named pUC-AAVR. This was used as a template to carry out PCR using oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 3 (5′-AAT[CCATGG]GCTCTGCAGGCGAGTCGGTTC-3′) and SEQ ID NO: 4 (5′-TTA[CTCGAG]TCAATGATGATGATGATGATGGTCTACCGCTTTGTTGACGG-3′) as

[0089] PCR primers (the square brackets in SEQ ID NO: 3 indicate the restriction enzyme NcoI recognition sequence, and the square brackets in SEQ ID NO: 4 indicate the restriction enzyme XhoI recognition sequence). Specifically, a reaction solution having the composition listed in Table 1 was prepared, the reaction solution was heat-treated at 98° C. for 5 minutes, and PCR was performed by repeating 30 cycles of a reaction including a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 90 seconds.

TABLE-US-00001 TABLE 1 Composition Volume Template DNA (10 ng/μL) 1 μL 10 μM PCR primer (SEQ ID NOS: 3, 33) 2 μL 10 μM PCR primer (SEQ ID NO: 4) 2 μL 5 × PrimeSTAR buffer 10 μL (manufactured by Takara Bio Inc.) 2.5 mM dNTPs 4 μL 2.5 U/μL PrimeSTAR HS 1 μL (manufactured by Takara Bio Inc.) H.sub.2O Up to 50 μL

[0090] (3) The polynucleotide obtained in (2) was purified and digested with restriction enzymes NcoI and XhoI, and ligated to an expression vector pET26b (manufactured by MERCK MILLIPORE), which was preliminarily digested with restriction enzymes NcoI and XhoI. The E. coli BL21 (DE3) strain was transformed using the ligation product.

[0091] (4) After the transformant obtained in (3) was cultured in LB (Luria-Bertani) medium containing 50 μg/mL kanamycin, a vector pET-AAVRD2 expressing the extracellular domains 1 and 2 of the AAV-binding protein was extracted using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0092] (5) The nucleotide sequence of the polynucleotide encoding the AAV-binding protein and its surrounding region in the expression vector pET-AAVRD2 prepared in (4) was analyzed by a fully automated DNA sequencer Genetic Analyzer3500 (manufactured by Thermo Fisher Scientific). In the analysis, an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 5 (5′-TAATACGACTCACTATAGGG-3′) or SEQ ID NO: 6 (5′-ATGCTAGTTATTGCTCAGCGG-3′) was used as a sequencing primer.

[0093] The amino acid sequence of a polypeptide expressed in the expression vector pET-AAVRD2 is set forth in SEQ ID NO: 7, and the sequence of a polynucleotide encoding the polypeptide is set forth in SEQ ID NO: 8. In SEQ ID NO: 7, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the extracellular domain of the AAV-binding protein (domains 1 and 2; the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219.

Example 2 Evaluation of AAV-Binding Ability of AAV-Binding Protein

[0094] (1) The E. coli BL21 (DE3) strain transformed with pET-AAVRD2 prepared in Example 1 was inoculated in 3 mL of 2YT liquid medium (peptone at 16 g/L, yeast extract at 10 g/L, sodium chloride at 5 g/L) containing 50 μg/mL kanamycin and aerobically cultured with shaking overnight at 37° C., thereby performing preculture.

[0095] (2) The preculture solution of (1) in a volume of 200 μL was inoculated in 20 mL of 2YT liquid medium supplemented with 50 μg/mL kanamycin in a 100 mL baffled flask and aerobically cultured with shaking at 37° C.

[0096] (3) The flask was cooled on ice 2.0 hours after the start of culture, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to yield a final concentration of 0.1 mM, and shaking culture was continued aerobically overnight at 25° C.

[0097] (4) After the end of culture, the culture solution in an amount of 2 mL was collected, and cells were collected by centrifugation. A protein extract containing the expressed AAV-binding protein was prepared using the obtained cells with a 200 μL of a BugBuster Protein extraction kit (manufactured by MERCK MILLIPORE). The prepared protein extract was diluted 30-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0098] (5) Preparation of VLP2 (virus-like particles of AAV2)

[0099] (5-1) The E. coli JM109 strain was transformed using pRC2-mi342 Vector (manufactured by Takara Bio Inc.) and pHelper Vector (manufactured by Takara Bio Inc.). The obtained transformants were cultured with shaking overnight at 37° C. in a 5 L baffled flask containing 1 L of 2YT medium supplemented with carbenicillin to yield a concentration of 100 μg/mL each.

[0100] (5-2) The cells were recovered by centrifuging the culture solution of (5-1). The pRC2-mi342 Vector and the pHelper Vector were prepared in large amounts from the obtained cells using Plasmid Mega Kits (manufactured by QIAGEN).

[0101] (5-3) HEK293T cells were cultured in five T-225 flasks (manufactured by Thermo Fisher Scientific) containing 40 mL of D-MEM medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) containing 10% (v/v) bovine serum. The pRC2-mi342 Vector and the pHelper Vector prepared in (5-2) were transfected into the cells using a TransIT-ViruGEN Transfection Reagent (manufactured by Takara Bio Inc.), and the cells were statically cultured for 3 days under the conditions of 5% carbon dioxide and 37° C.

[0102] (5-4) VLP2 was obtained by collecting the cells after the culture in (5-3) and performing extraction and purification with an AAVpro Purification Kit (manufactured by Takara Bio Inc.). About 1 mL of a purified VLP2 solution was prepared from the five T-225 flasks.

[0103] (6) The binding property of the AAV-binding protein in the protein extract prepared in (4) to VLP2 prepared in (5) was evaluated by the ELISA method.

[0104] (6-1) VLP2 prepared in (5) was diluted 200- to 1000-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and added to a 96-well microplate (manufactured by Thermo Fisher scientific) at 100 μL/well to be immobilized thereon (at 4° C. for 18 hours). After the end of immobilization, blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w/v) SKIM MILK (manufactured by Becton, Dickinson and Company) and 150 mM sodium chloride.

[0105] (6-2) After washing with washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w/v] Tween 20 (trade name) and 150 mM sodium chloride), a solution containing the AAV-binding protein to be evaluated for VLP2-binding activity was added such that the AAV-binding protein was reacted with VLP2 (at 30° C. for 1 hour).

[0106] (6-3) After the end of the reaction, the reaction product was washed with the washing buffer, and an Anti-6His antibody (manufactured by Bethyl Laboratories) diluted to 100 ng/mL was added at 100 μL/well.

[0107] (6-4) After the reaction at 30° C. for 1 hour, the reaction product was washed with the washing buffer, and TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL/well. Color development was stopped by adding 1M phosphoric acid at 50 μL/well. The absorbance at 450 nm was measured using a microplate reader (manufactured by Tecan Group Ltd.).

[0108] FIG. 1 shows the measurement results of the ELISA method. High absorption was observed when VLP2 was immobilized on the solid phase (VLP (+)), indicating that the AAV-binding protein expressed in this Example binds to VLP2. Since VLP2 is a virus-like particle of AAV2, this result can mean that the AAV-binding protein binds to AAV2.

Example 3 Creation and Screening of Mutation Library of AAV-Binding Proteins (Part 1)

[0109] The polynucleotide portion encoding the AAV-binding protein in the AAV-binding protein expression vector pET-AAVRD2 was subjected to random mutagenesis by error-prone PCR.

[0110] (1) Error-prone PCR was performed using pET-AAVRD2 prepared in Example 1 as a template. After a reaction solution having the composition listed in Table 2 was prepared, the reaction solution was heat-treated at 98° C. for 5 minutes, a reaction including a first step at 98° C. for 30 seconds, a second step at 55° C. for 20 seconds, and a third step at 72° C. for 90 seconds was repeated 30 cycles, and lastly, heat treatment was performed at 72° C. for 5 minutes. Thus, error-prone PCR was performed. Mutations were successfully introduced into the polynucleotide encoding the AAV-binding protein by the error-prone PCR, and the average mutation rate was 1.8 amino acid mutations per molecule.

TABLE-US-00002 TABLE 2 Composition Volume Template DNA (pET-AAVRD2) (10 ng/μL) 1 μL 10 μM PCR primer (SEQ ID NO: 3) 2 μL 10 μM PCR primer (SEQ ID NO: 4) 2 μL 10 mM MnCl.sub.2 1.5 μL 2.5 mM dNTPs 4 μL 10 × Ex Taq Buffer 5 μL (manufactured by Takara Bio Inc.) Go Taq polymerase 0.5 μL (manufactured by Promega Corporation) H.sub.2O up to 50 μL

[0111] (2) The PCR product obtained in (1) was purified and then digested with restriction enzymes NcoI and XhoI and ligated to an expression vector pET26b (manufactured by MERCK MILLIPORE), which was preliminarily digested with the same restriction enzymes.

[0112] (3) After the end of the ligation reaction, E. coli BL21 (DE3) was transformed with the reaction solution and cultured in LB plate medium containing 50 μg/mL kanamycin (at 37° C. for 18 hours). Subsequently, the colonies formed on the plate were used as a random mutant library.

[0113] (4) The random mutant library (transformants) prepared in (3) was inoculated in 200 μL of 2YT liquid medium containing 50 μg/mL kanamycin, and cultured with shaking overnight at 37° C. using a 96-well deep well plate.

[0114] (5) The culture solution prepared in (4) in an amount of 10 μL was subcultured in 500 μL of 2YT liquid medium containing 0.1 mM IPTG and 50 μg/mL kanamycin, and further cultured with shaking overnight at 25° C. using a 96-well deep well plate.

[0115] (6) The culture solution of (5) was centrifuged, and the obtained culture supernatant was diluted 2-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. The diluted solution was heat-treated at 43° C. for 15 minutes.

[0116] (7) The binding activity of AAV-binding protein to VLP2 when the heat treatment of (6) was performed and the binding activity of AAV-binding protein to VLP2 when the heat treatment of (6) was not performed were measured by the ELISA method described in Example 2 (6). The residual activity was calculated by dividing the binding activity of AAV-binding protein to VLP2 when the heat treatment was performed by the binding activity of AAV-binding protein to VLP2 when the heat treatment was not performed.

[0117] (8) About 2700 transformants were evaluated by the method of (7), and among them, transformants expressing AAV-binding proteins having improved heat stability as compared with the wild-type (without amino acid substitution) AAV-binding protein were selected. Each selected transformant was cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0118] (9) The nucleotide sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed by the same method as described in Example 1 (5), and the amino acid mutation site was identified.

[0119] Table 3 summarizes the amino acid substitution positions of the AAV-binding proteins expressed by the transformants selected in (8) above with respect to the wild-type (without amino acid substitution) AAV-binding protein and the residual activity (%) thereof after heat treatment. It can be said that an AAV-binding protein, in which amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 have at least one of the following amino acid substitutions, has improved heat stability as compared with the wild-type AAV-binding protein: Ile319Phe (this notation indicates a substitution of isoleucine at position 319 of SEQ ID NO: 1 with phenylalanine; the same applies hereinafter), Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, Asn329Tyr, Ala330Gly, Tyr331Cys, Val332Gln, Leu333Val, Leu333Pro, Gln334Arg, Pro337Leu, Lys338Arg, Lys338Glu, Glu340Gly, Thr341Ala, Tyr342Cys, Tyr342His, Tyr342Asn, Thr343Met, Tyr344His, Asp345Asn, Trp346Leu, Trp346Arg, Gln347Leu, Gln347Pro, Leu348Pro, Ile349Thr, Thr350Met, His351Leu, Pro352Leu, Arg353Cys, Asp354Gly, Tyr355His, Tyr355Asn, Tyr355Cys, Ser356Cys, Gly357Cys, His363Arg, His363Leu, Ser364Pro, Gln365Arg, Ile366Thr, Leu367Pro, Lys368Arg, Leu369Gln, Leu369Pro, Ser370Ala, Lys371Glu, Thr373Ala, Leu376Pro, Tyr377Cys, Phe379Ser, Val381Ala, Glu384Gly, Asn387Ser, His389Gln, His389Leu, His389Arg, Gly390Ser, Glu391Lys, Tyr393Cys, Val396Ala, Lys399Glu, Lys399Arg, Arg406Ser, Val412Ala, Gln415Leu, Phe416Ser, Gln441Leu, Tyr442Phe, Lys448Arg, Glu453Gly, Lys455Arg, Glu458Gly, Ala461Ser, Ser476Arg, Leu477Pro, Asn487Asp, and Asn492Asp.

TABLE-US-00003 TABLE 3 Residual Amino acid activity No. substitution [%] 1 Asn324Asp 46.0 2 Tyr331Cys 73.4 3 Lys338Glu 52.6 4 Tyr344His 88.2 5 Asp345Asn 71.4 6 Trp346Leu 85.7 7 Leu348Pro 69.6 8 His351Leu 68.7 9 Pro352Leu 71.9 10 Arg353Cys 84.3 11 Tyr355Asn 80.6 12 Ser356Cys 84.6 13 Gly357Cys 97.0 14 Gln365Arg 57.7 15 Leu367Pro 56.9 16 Leu369Gln 92.8 17 Phe379Ser 80.9 18 Tyr393Cys 86.4 19 Val396Ala 62.3 20 Lys399Glu 63.3 21 Tyr442Phe 48.7 22 Leu477Pro 79.9 23 Asn487Asp 58.3 24 Asn492Asp 63.2 25 Asn324Asp, Tyr355His 77.6 26 Asn329Tyr, Lys399Arg 61.9 27 Tyr331Cys, Leu376Pro 74.4 28 Tyr331Cys, Glu384Gly 83.7 Wild type 43.4 29 Leu333Val, Tyr342Cys 91.8 30 Glu340Gly, Tyr342Cys 91.3 31 Tyr342His, Ile349Thr 66.8 32 Tyr342Cys, Gly390Ser 97.6 33 Tyr344His, His363Arg 82.2 34 Trp346Arg, Glu458Gly 78.1 35 Gln347Leu, Thr373Ala 85.9 36 Tyr355His, Asn387Ser 86.6 37 Gln365Arg, His389Gln 70.1 38 Gln365Arg, Gln415Leu 65.3 39 Ile366Thr, Gln441Leu 65.5 40 Phe379Ser, Lys455Arg 77.4 41 Glu453Gly, Ser476Arg 56.1 42 Ile319Phe, Pro322Leu, Lys448Arg 56.6 43 Leu321Pro, Tyr342Asn, Ala461Ser 84.1 44 Gln327Arg, Lys338Arg, Leu348Pro 76.1 45 Asn329Asp, Tyr342His, Tyr355Cys 97.2 46 Asn329Asp, Ile366Thr, Lys368Arg 49.3 47 Asn329Tyr, Tyr377Cys, Arg406Ser 92.7 48 Ala330Gly, Gln347Pro, Lys371Glu 88.4 49 Tyr331Cys, Thr343Met, Tyr355Cys 97.7 50 Val332Gln, Tyr342Cys, Val412Ala 80.3 51 Leu333Pro, Pro337Leu, Thr350Met 66.7 52 Leu348Pro, Leu376Pro, Val396Ala 80.9 53 Asp354Gly, Leu369Gln, Lys455Arg 90.5 54 His363Leu, Ser364Pro, His389Leu 87.2 55 Gln365Arg, Ser370Ala, Glu391Lys 65.7 56 Asn324Ser, Thr341Ala, Leu369Pro, Phe416Ser 81.3 57 Gln334Arg, Val381Ala, His389Arg, Tyr393Cys 79.0

Example 4 Evaluation of Thermal Stability of AAV-Binding Protein Mutants (Amino Acid Substitution Products)

[0120] Among the amino acid-substituted (mutated) AAV-binding proteins listed in Table 3, No. 9 (Pro352Leu substitution product), No. 16 (Leu369Gln substitution product), No. 29 (Leu333 Val-Tyr342Cys substitution product), No. 30 (Glu340Gly-Tyr342Cys substitution product), No. 32 (Tyr342Cys-Gly390Ser substitution product), No. 36 (Tyr355His-Asn387Ser substitution product), No. 43 (Leu321Pro-Tyr342Asn-Ala461Ser substitution product), No. 45 (Asn329Asp-Tyr342Hi s-Tyr355Cys substitution product), No. 47 (Asn329Tyr-Tyr377Cys-Arg406Ser substitution product), No. 49 (Tyr331Cys-Thr343Met-Tyr355Cys substitution product), and No. 53 (Asp354Gly-Leu369Gln-Lys455Arg substitution product) were selected, and the heat stability was evaluated again.

[0121] The nucleotide sequence and amino acid sequence of No. 9 are set forth in SEQ ID NOS: 9 and 10, respectively.

The nucleotide sequence and amino acid sequence of No. 16 are set forth in SEQ ID NOS: 11 and 12, respectively.
The nucleotide sequence and amino acid sequence of No. 29 are set forth in SEQ ID NOS: 13 and 14, respectively.
The nucleotide sequence and amino acid sequence of No. 30 are set forth in SEQ ID NOS: 15 and 16, respectively.
The nucleotide sequence and amino acid sequence of No. 32 are set forth in SEQ ID NOS: 17 and 18, respectively.
The nucleotide sequence and amino acid sequence of No. 36 are set forth in SEQ ID NOS: 19 and 20, respectively.
The nucleotide sequence and amino acid sequence of No. 43 are set forth in SEQ ID NOS: 21 and 22, respectively.
The nucleotide sequence and amino acid sequence of No. 45 are set forth in SEQ ID NOS: 23 and 24, respectively.
The nucleotide sequence and amino acid sequence of No. 47 are set forth in SEQ ID NOS: 25 and 26, respectively.
The nucleotide sequence and amino acid sequence of No. 49 are set forth in SEQ ID NOS: 27 and 28, respectively.
The nucleotide sequence and amino acid sequence of No. 53 are set forth in SEQ ID NOS: 29 and 30, respectively.

[0122] In addition, the Pro352Leu substitution in the amino acid sequence of No. 9 (SEQ ID NO: 10) is located at position 65 in SEQ ID NO: 10.

The Leu369Gln substitution in the amino acid sequence of No. 16 (SEQ ID NO: 12) is located at position 82 in SEQ ID NO: 12.
The Leu333Val and Tyr342Cys substitutions in the amino acid sequence of No. 29 (SEQ ID NO: 14) are located at positions 46 and 55 in SEQ ID NO: 14, respectively.
The Glu340Gly and Tyr342Cys substitutions in the amino acid sequence of No. 30 (SEQ ID NO: 16) are located at positions 53 and 55 in SEQ ID NO: 16, respectively.
The Tyr342Cys and Gly390Ser substitutions in the amino acid sequence of No. 32 (SEQ ID NO: 18) are located at positions 55 and 103 in SEQ ID NO: 18, respectively.
The Tyr355His and Asn387Ser substitutions in the amino acid sequence of No. 36 (SEQ ID NO: 20) are located at positions 68 and 100 in SEQ ID NO: 20, respectively.
The Leu321Pro, Tyr342Asn, and Ala461Ser substitutions in the amino acid sequence of No. 43 (SEQ ID NO: 22) are located at positions 34, 55, and 174 in SEQ ID NO: 22, respectively.
The Asn329Asp, Tyr342His, and Tyr355Cys substitutions in the amino acid sequence of No. 45 (SEQ ID NO: 24) are located at positions 42, 55, and 68 in SEQ ID NO: 24, respectively.
The Asn329Tyr, Tyr377Cys, and Arg406Ser substitutions in the amino acid sequence of No. 47 (SEQ ID NO: 26) are located at positions 42, 90, and 119 in SEQ ID NO: 26, respectively.
The Tyr331Cys, Thr343Met, and Tyr355Cys substitutions in the amino acid sequence of No. 49 (SEQ ID NO: 28) are located at positions 44, 56, and 68 in SEQ ID NO: 28, respectively.
The Asp354Gly, Leu369Gln, and Lys455Arg substitutions in the amino acid sequence of No. 53 (SEQ ID NO: 30) are located at positions 67, 82, and 168 in SEQ ID NO: 30, respectively.

[0123] (1) The transformants expressing 11 types of mutated AAV-binding proteins selected above and the transformant expressing the wild-type AAV-binding protein obtained in Example 1 (3) were each cultured by the methods described in Examples 3 (4) and (5), and then the culture solutions were centrifuged, thereby obtaining culture supernatants containing the expressed mutated AAV-binding proteins and a culture supernatant containing the wild-type AAV-binding protein.

[0124] (2) The binding activity of the AAV-binding protein in each culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, each culture supernatant obtained in (1) was diluted with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride such that the measured values would be the same.

[0125] (3) The diluted protein solution was divided into three portions, and two of them were heat-treated at 50.3° C. or 66.1° C. for 15 minutes using a thermal cycler (manufactured by Eppendorf SE) while the remaining one was not heat-treated.

[0126] (4) The binding activity between the heat-treated or non-heat treated AAV-binding activity protein of (3) and VLP2 was measured by the ELISA method described in Example 2 (6), and the residual activity was calculated by dividing the absorbance at 450 nm with heat treatment by the absorbance at 450 nm without heat treatment.

[0127] Table 4 shows the results. It was confirmed that all of the mutated AAV-binding proteins had higher residual activity as compared with the wild-type AAV-binding protein, and therefore, the heat stability of the mutated AAV-binding protein was improved.

TABLE-US-00004 TABLE 4 AAV-binding protein Residual activity [%] No. Amino acid substitution 50.3° C. 66.1° C. 9 Pro352Leu 49.0 46.5 16 Leu369Gln 32.0 20.3 29 Leu333Val, Tyr342Cys 66.7 54.5 30 Glu340Gly, Tyr342Cys 65.4 52.4 32 Tyr342Cys, Gly390Ser 76.0 70.5 36 Tyr355His, Asn387Ser 74.5 51.0 43 Leu321Pro, Tyr342Asn, Ala461Ser 55.6 56.3 45 Asn329Asp, Tyr342His, Tyr355Cys 66.0 46.7 47 Asn329Tyr, Tyr377Cys, Arg406Ser 64.5 58.7 49 Tyr331Cys, Thr343Met, Tyr355Cys 77.3 57.3 53 Asp354Gly, Leu369Gln, Lys455Arg 29.8 23.5 Wild type 16.1 9.6

Example 5 Evaluation of Acid Stability of Mutated AAV-Binding Protein (Part 1)

[0128] (1) The transformants expressing the mutated AAV-binding proteins evaluated in Example 4 (i.e., mutated AAV-binding proteins according to Nos. 9, 16, 29, 30, 32, 36, 43, 45, 47, 49, and 53 in Table 3) and the transformant expressing the wild-type AAV-binding protein obtained in Example 1 (3) were each cultured by the methods described in Examples 3 (4) and (5), and then the culture solutions were centrifuged, thereby obtaining culture supernatants containing the expressed mutated AAV-binding proteins or a culture supernatant containing the wild-type AAV-binding protein.

[0129] (2) The binding activity of the AAV-binding protein in each culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, each culture supernatant obtained in (1) was diluted with pure water such that the measured values would be the same.

[0130] (3) The diluted protein solution was divided into two portions, one of which was mixed with an equal amount of 0.1 M glycine-HCl buffer (pH 2.5). After standing still at 30° C. for 2 hours and 24 hours, the pH was adjusted to around 6 by mixing the solution with 0.5 M IVIES buffer (pH 6.0) at a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6). The other one was not acid-treated, and the dilution ratio was the same as the condition of the acid treatment case. The pH was adjusted to around 6 by mixing the solution with 0.5 M IVIES buffer (pH 6.0) at a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6).

[0131] (4) The residual activity was calculated by dividing the absorbance at 450 nm with the acid treatment described in (3) by the absorbance at 450 nm without the acid treatment.

[0132] Table 5 shows the results. In all the mutated AAV-binding proteins evaluated in Example 4, the residual activity after treatment for 2 hours and 24 hours under the conditions of 30° C. and pH 2.5 was improved as compared with the wild type. In other words, it was confirmed that the acid stability of the mutated AAV-binding proteins was improved. In particular, No. 30 (Glu340Gly-Tyr342Cys substitution product, SEA ID NO: 16), No. 32 (Tyr342Cys-Gly390Ser substitution product, SEQ ID NO:18), and No. 45 (Asn329Asp-Tyr342His-Tyr355Cys substitution product, SEQ ID NO:24) had significantly improved acid stability as compared with the wild-type AAV-binding protein.

TABLE-US-00005 TABLE 5 PH 2.5 AAV-binding protein Residual activity [%] No. Amino acid substitution 2 h 24 h 9 Pro352Leu 17.6 5.6 16 Leu369Gln 17.3 10.0 29 Leu333Val, Tyr342Cys 52.2 40.2 30 Glu340Gly, Tyr342Cys 70.5 56.1 32 Tyr342Cys, Gly390Ser 70.7 64.7 36 Tyr355His, Asn387Ser 16.1 8.9 43 Leu321Pro, Tyr342Asn, Ala461Ser 41.4 28.3 45 Asn329Asp, Tyr342His, Tyr355Cys 64.4 47.6 47 Asn329Tyr, Tyr377Cys, Arg406Ser 43.6 34.8 49 Tyr331Cys, Thr343Met, Tyr355Cys 52.8 16.6 53 Asp354Gly, Leu369Gln, Lys455Arg 18.2 13.2 Wild type 7.9 5.1

Example 6 Creation and Screening of Mutation Library of AAV-Binding Proteins (Part 2)

[0133] Among the mutated AAV-binding proteins evaluated in Examples 4 and 5, No. 32 (Tyr342Cys-Gly390Ser substitution product, SEQ ID NO: 18) was selected (named AVR2). The polynucleotide portion encoding the protein was subjected to random mutagenesis by error-prone PCR.

[0134] (1) Using the plasmid pET-AVR2 into which the polynucleotide (SEQ ID NO: 17) encoding AVR2 (SEQ ID NO: 18) was inserted as a template, a reaction solution having the composition listed in Table 6 was prepared, and then error-prone PCR was performed under the same temperature conditions as in Example 3 (1). Mutations were successfully introduced into the polynucleotide encoding AVR2 by the error-prone PCR, and the average mutation rate was 1.5 amino acid mutations per molecule.

TABLE-US-00006 TABLE 6 Composition Volume 10 ng/μL template DNA 1 μL 10 μM PCR primer (SEQ ID NO: 3) 2 μL 10 μM PCR primer (SEQ ID NO: 4) 2 μL 10 mM MnCl.sub.2 0.5 μL 2.5 mM dNTPs 4 μL 10 × Ex Taq Buffer (manufactured by Takara 5 μL Bio Inc.) Go Taq polymerase (manufactured by Promega 0.5 μL Corporation) H.sub.2O up to 50 μL

[0135] (2) The PCR product obtained in (1) was purified and then digested with restriction enzymes NcoI and XhoI and ligated to an expression vector pET26b (manufactured by MERCK MILLIPORE), which was preliminarily digested with the same restriction enzymes.

[0136] (3) After the end of the ligation reaction, E. coli BL21 (DE3) was transformed with the reaction solution and cultured in LB plate medium containing 50 μg/mL kanamycin (at 37° C. overnight). Subsequently, the colonies formed on the plate were used as a random mutant library.

[0137] (4) The random mutant library (transformants) prepared in (3) cultured by the methods described in Example 3 (4) and (5).

[0138] (5) The culture solution of (4) was centrifuged, and the obtained culture supernatant was diluted 4-fold with ultrapure water. The diluted culture solution in an amount of 60 μL and 60 μL of 0.1 M glycine-HCl buffer (pH 3.0) were mixed, and heat treatment and acid treatment were performed at 51.1° C. for 15 minutes.

[0139] (6) The binding activity of AAV-binding protein to VLP2 when the heat treatment of (5) was performed and the binding activity of AAV-binding protein to VLP2 when the heat treatment of (5) was not performed were measured by the ELISA method described in Example 2 (6). The residual activity was calculated by dividing the binding activity of AAV-binding protein to VLP2 when the heat treatment was performed by the binding activity of AAV-binding protein to VLP2 when the heat treatment was not performed.

[0140] (7) A random mutant library of about 1800 transformants was evaluated by the method of (6), and among them, transformants expressing AAV-binding proteins having improved residual activity as compared with the parent molecule AVR2 were selected. Each selected transformant was cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0141] (8) The nucleotide sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and the amino acid mutation site was identified.

[0142] Table 7 summarizes the amino acid substitution positions of the AAV-binding proteins expressed by the transformants selected in (7) above with respect to AVR2 (Tyr342Cys-Gly390Ser substitution product) and the residual activity (%) thereof after heat treatment and acid treatment.

[0143] It can be said that an AAV-binding protein, in which amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 have at least one of the following amino acid substitutions, has improved heat stability and acid stability as compared with the wild-type AAV-binding protein: Ile319Phe, Ile319Asn, Ile319Ser, Thr320Ile, Leu321Pro, Lys323Glu, Leu328Gln, Leu328Pro, Tyr331His, Val332Ala, Val332Glu, Leu333Pro, Pro336Gln, Pro337Gln, Lys338Asn, Tyr(Cys)342Arg (this notation indicates a substitution of tyrosine at position 342 of SEQ ID NO: 1 with cysteine and then further by arginine; the same applies hereinafter), Thr350Ser, Gln365Arg, Ile366Phe, Phe379Ser, Val383Ala, Gln386Arg, His389Asp, Gly392Cys, Val394Ala, Val396Ala, Lys399Glu, Ile409Val, Ser476Gly, Ser476Arg, Asn487Asp, Thr490Ser, and Asn492Asp.

TABLE-US-00007 TABLE 7 No. Amino acid substitution Residual activity [%] No. Amino acid substitution Residual activity [%] 58 Ile319Phe 73.9 71 His389Asp 71.9 59 Ile319Asn 26.1 72 Val394Ala 32.0 60 Lys323Glu 80.2 73 Val396Ala 48.9 61 Leu328Gln 43.5 74 Ser476Gly 63.4 62 Leu328Pro 38.8 75 Ser476Arg 96.3 63 Tyr331His 43.1 76 Asn492Asp 36.4 64 Val332Ala 41.4 77 Leu321Pro, Ile409Val 34.3 65 Val332Glu 29.4 78 Leu328Pro, Ile366Phe 55.9 66 Leu333Pro 31.8 79 Lys338Asn, Asn487Asp 73.1 67 Pro337Gln 45.6 80 Gln365Arg, Gly392Cys 49.9 68 Tyr(Cys)342Arg 70.4 81 Phe379Ser, Ser476Gly 66.3 69 Thr350Ser 49.9 82 Ser476Arg, Thr490Ser 70.6 70 Val383Ala 32.8 83 Ile319Ser, Thr320Ile, Lys399Glu 70.2 32 AVR2 25.5 84 Pro336Gln, Gln386Arg, Asn487Asp 48.4

Example 7 Integration of Stabilized Amino Acid Substitutions (Part 1)

[0144] By integrating the amino acid substitutions involved in the improvement of heat stability and acid stability of the AAV-binding proteins found in Example 6 in AVR2 (SEQ ID NO: 18), the stability was further improved. Specifically, among the amino acid-substituted (mutated) AAV-binding proteins listed in Table 7, No. 75 (integrated amino acid substitutions of Ser476Arg for AVR2) was selected (named AVR3). The amino acid substitutions described in any one of the following (a) to (d) were integrated into the protein. The Ser476Arg substitution in the amino acid sequence of AVR3 (SEQ ID NO: 32) is located at position 189 in SEQ ID NO: 32.

(a) Ile319Phe (named AVR4a)
(b) Asn487Asp (named AVR4d)
(c) Ile319Phe and Lys323Glu (named AVR5b)
(d) Lys399Glu and Asn487Asp (named AVR5e)

[0145] Hereinafter, a method for producing the following AAV-binding proteins will be described in detail.

[0146] (a) AVR4a

[0147] From the amino acid substitutions clarified in Example 6 that are involved in improving the stability to heat and acid, Ile319Phe was selected and integrated in AVR3 (SEQ ID NO: 32), thereby preparing AVR4a. Specifically, AVR4a was prepared by introducing a mutation that yields Ile319Phe in the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32).

[0148] (a-1) A reaction solution having the composition listed in Table 1 was prepared using the plasmid pET-AVR3 into which the polynucleotide (SEQ ID NO: 31) encoding AVR3 was inserted as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 33 (5′-CCATGGGCTCTGCAGGCGAGTCGGTTCAGTTTACCC-3′) as PCR primers. The reaction solution was heat-treated at 98° C. for 5 minutes, a reaction including a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 1 minute was repeated 30 cycles, and lastly, heat treatment was performed at 72° C. for 5 minutes. Thus, PCR was performed.

[0149] (a-2) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN). The purified PCR product was named 4ap.

[0150] (a-3) 4ap obtained in (a-2) was purified and digested with restriction enzymes NcoI and XhoI, and ligated to an expression vector pET26b (manufactured by MERCK MILLIPORE), which was preliminarily digested with restriction enzymes NcoI and XhoI. The E. coli BL21 (DE3) strain was transformed using the ligation product.

[0151] (a-4) The transformant obtained in (a-3) was cultured in LB medium supplemented with 50 μg/mL kanamycin. Plasmids were extracted from the collected bacterial cells (transformants), thereby obtaining a plasmid pET-AVR4a containing a polynucleotide encoding AVR4a with four amino acid substitutions in the wild-type AAV-binding protein.

[0152] (a-5) The nucleotide sequence of pET-AVR4a was analyzed in the same manner as in Example 1 (5).

[0153] The amino acid sequence of AVR4a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 34, and the sequence of the polynucleotide encoding AVR4a is set forth in SEQ ID NO: 35. In SEQ ID NO: 34, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR4a (domains 1 and 2 corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 34, phenylalanine of Ile319Phe is present at position 32, cysteine of Tyr342Cys is present at position 55, serine of Gly390Ser is present at position 103, and arginine of Ser476Arg is present at position 189.

[0154] (b) AVR4d

[0155] From the amino acid substitutions clarified in Example 6 that are involved in improving the stability to heat and acid, Asn487Asp was selected and accumulated in AVR3 (SEQ ID NO: 32), thereby preparing AVR4d. Specifically, AVR4d was prepared by introducing a mutation that causes Asn487Asp in the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32).

[0156] (b-1) A reaction solution having the composition listed in Table 8 was prepared using pET-AVR3 as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 36 (5′-GATTCTGATGGCGCAACCGACTCCACCACC-3′) as PCR primers. The reaction solution was heat-treated at 98° C. for 5 minutes, a reaction including a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 1 minute was repeated 30 cycles, and lastly, heat treatment was performed at 72° C. for 5 minutes. Thus, PCR was performed.

TABLE-US-00008 TABLE 8 Composition Volume 10 ng/μL template DNA 1 μL 10 μM PCR primer (SEQ ID NOS: 5, 37, 40, 44) 1 μL 10 μM PCR primer (SEQ ID NOS: 36, 6, 41, 45) 1 μL 5 × PrimeSTAR Buffer (manufactured by Takara 10 μL Bio Inc.) 2.5 mM dNTPs 4 μL 2.5 U/μL PrimeSTAR HS (manufactured by Takara 1 μL Bio Inc.) H.sub.2O up to 50 μL

[0157] (b-2) The amplified PCR product was purified by the same method as in (a-2). The purified PCR product was named 4dF.

[0158] (b-3) PCR was performed in the same manner as in (b-1) and (b-2) except that pET-AVR3 was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 37 (5′-GGTGGTGGAGTCGGTTGCGCCATCAGAATC-3′) were used as PCR primers. The purified PCR product was named 4dR.

[0159] (b-4) The two types of PCR products (4dF, 4dR) obtained in (b-2) and (b-3) were mixed, thereby preparing a reaction solution having the composition listed in Table 9. The reaction solution was heat-treated at 98° C. for 5 minutes, and PCR was performed by a reaction of 30 cycles of a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 1 minute, thereby obtaining a PCR product 4 dp in which 4dF and 4dR were joined to each other.

TABLE-US-00009 TABLE 9 Composition Volume PCR product (4dF) 1 μL PCR product (4dR) 1 μL 10 μM PCR primer (SEQ ID NO: 5) 1 μL 10 μM PCR primer (SEQ ID NO: 6) 1 μL 5 × PrimeSTAR Buffer (manufactured by Takara 10 μL Bio Inc.) 2.5 mM dNTPs 4 μL 2.5 U/μL PrimeSTAR HS (manufactured by Takara 1 μL Bio Inc.) H.sub.2O up to 50 μL

[0160] (b-5) The PCR product 4 dp obtained in (b-4) was treated in the same manner as in (a-3) and (a-4), thereby obtaining a plasmid pET-AVR4d containing a polynucleotide encoding AVR4d with four amino acid substitutions in the wild-type AAV-binding protein.

[0161] (b-6) The nucleotide sequence of pET-AVR4d was analyzed in the same manner as in Example 1 (5).

[0162] The amino acid sequence of AVR4d with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 38, and the sequence of the polynucleotide encoding AVR4d is set forth in SEQ ID NO: 39. In SEQ ID NO: 38, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR4d (corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 38, cysteine of Tyr342Cys is present at position 55, serine of Gly390Ser is present at position 103, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

[0163] (c) AVR5b

[0164] From the amino acid substitutions clarified in Example 6 that are involved in improving the stability to heat and acid, Ile319Phe and Lys323Glu were selected and integrated in AVR3 (SEQ ID NO: 32), thereby preparing AVR5b. Specifically, AVR5b was prepared by introducing mutations that yield Ile319Phe and Lys323Glu in the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32).

[0165] (c-1) A reaction solution having the composition listed in Table 8 was prepared using pET-AVR3 as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 40 (5′-TTTT[GGTCTC]AGTTCTCCGGCAGGGTAACTGAACCGAC-3′) and SEQ ID NO: 41 (5′-TTTT[GGTCTC]AGAACGAAGTACAACTGAATGCGTATGTG-3′) (both the square brackets in SEQ ID NO: 40 and the square brackets in SEQ ID NO: 41 indicate the restriction enzyme BsaI recognition sequence) as PCR primers. The reaction solution was heat-treated at 98° C. for 5 minutes, a reaction including a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 6 minutes was repeated 30 cycles, and lastly, heat treatment was performed at 72° C. for 5 minutes. Thus, PCR was performed.

[0166] (c-2) The amplified PCR product was purified by the same method as in (a-2). The purified PCR product was named 5 bp.

[0167] (C-3) The 5 bp obtained in (c-2) was treated with the restriction enzyme DpnI (manufactured by NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The thus obtained gene was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

[0168] (c-4) The DpnI-treated product obtained in (c-3) was digested with a restriction enzyme BsaI (manufactured by NEB) under the reaction solution having the composition listed in Table 10 and ligated with T4DNA ligase (manufactured by NEB).

TABLE-US-00010 TABLE 10 Composition Volume PCR product (20 ng/μL) 5 μL 10 × CutSmart Buffer (manufactured by NEB) 1.5 μL BsaI (manufactured by NEB) 1 μL T4 DNA ligase buffer (manufactured by NEB) 1.5 μL T4 DNA ligase (manufactured by NEB) 1 μL H.sub.2O up to 15 μL

[0169] (c-5) The E. coli BL21 (DE3) strain was transformed using the ligation product of (c-4). The obtained transformant was cultured in LB medium supplemented with 50 μg/mL kanamycin. Plasmids were extracted from the collected bacterial cells (transformants), thereby obtaining a plasmid pET-AVR5b containing a polynucleotide encoding AVR5b with five amino acid substitutions in the wild-type AAV-binding protein.

[0170] (c-6) The nucleotide sequence of pET-AVR5b was analyzed in the same manner as in Example 1 (5).

[0171] The amino acid sequence of AVR5b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 42, and the sequence of the polynucleotide encoding AVR5b is set forth in SEQ ID NO: 43. In SEQ ID NO: 42, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR5b (corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 42, phenylalanine of Ile319Phe is present at position 32, glutamic acid of Lys323Glu is present at position 36, cysteine of Tyr342Cys is present at position 55, serine of Gly390Ser is present at position 103, and arginine of Ser476Arg is present at position 189.

[0172] (d) AVR5e

[0173] From the amino acid substitutions clarified in Example 6 that are involved in improving the stability to heat and acid, Lys399Glu and Asn487Asp were selected and integrated in AVR3 (SEQ ID NO: 32), thereby preparing AVR5e. Specifically, AVR5e was prepared by introducing a mutation that yields Lys399Glu in the polynucleotide (SEQ ID NO: 39) encoding AVR4d (SEQ ID NO: 38) prepared in (b).

[0174] (d-1) A plasmid pET-AVR5e containing the polynucleotide encoding AVR5e with five amino acid substitutions in the wild-type AAV-binding protein was obtained in the same manner as in (c-1) to (c-5) except that pET-AVR4d obtained in (b-5) was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 44 (5′-TTTT[GGTCTC]ACTTGCGTGGCTCCGGTTCCACGGTAACG-3′) and SEQ ID NO: 45 (5′-TTTT[GGTCTC]ACAAGAACCGTCCTCCGATCGCCATTG-3′) (both the square brackets in SEQ ID NO: 44 and the square brackets in SEQ ID NO: 45 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0175] (d-2) The nucleotide sequence of pET-AVR5e was analyzed in the same manner as in Example 1 (5).

[0176] The amino acid sequence of AVR5e with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 46, and the sequence of the polynucleotide encoding AVR5e is set forth in SEQ ID NO: 47. In SEQ ID NO: 46, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR5e (corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 46, cysteine of Tyr342Cys is present at position 55, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

Example 8 Evaluation of Acid Stability of Mutated AAV-Binding Protein (Part 2)

[0177] (1) The transformants expressing the mutated AAV-binding protein AVR3 obtained in Example 6 and the mutated AAV-binding proteins (AVR4a, AVR4d, AVR5b, and AVR5e) prepared in Example 7 were each cultured by the methods described in Examples 3 (4) and (5), and then the culture solutions were centrifuged, thereby obtaining culture supernatants containing the expressed mutated AAV-binding proteins.

[0178] (2) The binding activity of the mutated AAV-binding protein in each culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, each culture supernatant obtained in (1) was diluted with pure water such that the measured values would be the same.

[0179] (3) Each diluted mutated AAV-binding protein solution was divided into four portions, and each portion was mixed with an equal amount of 0.1 M glycine-HCl buffer (pH 3.0). Three of the four samples were heat-treated at 51.5° C., 60.1° C., and 66.1° C., respectively, for 15 minutes, and the remaining one was left to stand still at room temperature (25° C.) for 15 minutes. Thereafter, the pH was adjusted to around 6 by mixing each sample with 0.5 M IVIES buffer (pH 6.0) at a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6).

[0180] (4) The residual activity was calculated by dividing the absorbance at 450 nm when the heat treatment and acid treatment described in (3) were performed by the absorbance at 450 nm when standing still was performed at room temperature.

[0181] Table 11 shows the results. Each of the mutated AAV-binding proteins (AVR3, AVR4a, AVR4d, AVR5b, and AVR5e) prepared in Example 7 had a residual activity higher than that of AVR3 (No. 75 in Table 7) regardless of the treatment temperature. This clarifies that the five mutated AAV-binding proteins have significantly improved stability to heat treatment under acidic conditions, i.e., stability to acid and heat, as compared with the wild-type AAV-binding protein.

TABLE-US-00011 TABLE 11 Residual activity after AAV-binding protein heat treatment [%] SEQ ID NO Name Amino acid substitution 51.5° C. 60.1° C. 66.1° C. 34 AVR4a Ile319Phe, Tyr342Cys, Gly390Ser, Ser476Arg 57 16 8 38 AVR4d Tyr342Cys, Gly390Ser, Ser476Arg, Asn487Asp 66 35 18 42 AVR5b Ile319Phe, Lys323Glu, Tyr342Cys, Gly390Ser, Ser476Arg 87 43 23 46 AVR5e Tyr342Cys, Gly390Ser, Lys399Glu, Ser476Arg, Asn487Asp 77 56 43 32 AVR3 Tyr342Cys, Gly390Ser, Ser476Arg 21 3 2

Example 9 Preparation of Mutated AAV-Binding Protein with Improved C-Terminus

[0182] (1) A reaction solution having the composition listed in Table 12 was prepared using pET-AVR5e prepared in Example 7 (d) as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 48 (5′-AT[CTCGAG]TCATCCGCAGGTATCGTTGCGGCAATGATGATGATGATGATGGTCTAC-3′) (the square brackets in SEQ ID NO: 48 indicate the restriction enzyme XhoI recognition sequence) as PCR primers. The reaction solution was heat-treated at 94° C. for 2 minutes, a reaction including a first step at 98° C. for 10 seconds, a second step at 52° C. for 30 seconds, and a third step at 68° C. for 1.5 minutes was repeated 25 cycles. Thus, PCR was performed.

TABLE-US-00012 TABLE 12 Composition Volume 10 ng/μL template 1 μL 10 μM PCR primer (SEQ ID NOS: 3, 57, 61, 67, 71, 72, 1.5 μL 74) 10 μM PCR primer (SEQ ID NOS: 48, 58, 62, 68, 73, 75) 1.5 μL 10 × KOD Buffer (manufactured by TOYOBO CO., LTD.) 5 μL 2 mM dNTPs 5 μL 25 mM MgSO.sub.4 3 μL 1 U/μL KOD Plus (manufactured by TOYOBO CO., LTD.) 1 μL H.sub.2O up to 50 μL

[0183] (1) The PCR product obtained in (1) was subjected to agarose gel electrophoresis, purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN), and digested with restriction enzymes NcoI and XhoI, and ligated to an expression vector pET26b (manufactured by MERCK MILLIPORE), which was preliminarily digested with restriction enzymes NcoI and XhoI. The E. coli BL21 (DE3) strain was transformed using the ligation product.

[0184] (3) After the obtained transformant was cultured in LB medium containing 50 μg/mL kanamycin, a plasmid pET-AVR5eHC encoding a protein (named AVR5eHC) having a tag for immobilization at the C-terminus of AVR5e was obtained using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0185] (4) The nucleotide sequence of pET-AVR5eHC obtained was analyzed in the same manner as in Example 1 (5).

[0186] The nucleotide sequence and amino acid sequence of AVR5eHC are set forth in SEQ ID NOS: 49 and 50, respectively. In SEQ ID NO: 50, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR5e ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, the histidine tag ranges from histidine (His) at position 214 to histidine (His) at position 219, and the cysteine tag sequence as a tag for immobilization ranges from cysteine (Cys) at position 220 to glycine (Gly) at position 226.

Example 10 Mass-Preparation of Mutated AAV-Binding Protein with Improved C-Terminus

[0187] (1) A transformant capable of expressing AVR5eHC obtained by transforming the E. coli BL21 (DE3) strain with pET-AVR5eHC prepared in Example 9 (3) was inoculated in a 200-mL baffled flask containing 40 mL of liquid medium (Phytone Peptone at 16 g/L, yeast extract at 10 g/L, sodium chloride at 5 g/L) containing 50 μg/mL kanamycin and aerobically cultured with shaking at 37° C. for 10 hours, thereby performing preculture.

[0188] (2) After inoculating 10 mL of the preculture solution of (1) in a 3-L fermenter (manufactured by Biott Corporation) into which 0.9 L of liquid medium containing glucose at 20 g/L, yeast extract at 40 g/L, trisodium phosphate dodecahydrate at 6 g/L, disodium hydrogen phosphate dodecahydrate at 18 g/L, ammonium chloride at 2 g/L, magnesium sulfate at 2 g/L, iron sulphate at 2 g/L, manganese chloride at 10 g/L, and kanamycin sulfate at 50 mg/L was introduced, the main culture was started under the conditions of a preset temperature of 30° C., a pH of 6.9 to 7.1, and an airflow rate of 3 VVM. For pH control, 50% (v/v) phosphoric acid is used as acid and 14% (w/v) ammonia water is used as alkali, and for the control of dissolved oxygen, the number of rotations of stirring was changed in the range of a lower limit of 500 rpm/an upper limit of 1000 rpm. At a time point when the glucose concentration became unmeasurable after the start of culture, a feed medium (glucose at 425 g/L, yeast extract at 140 g/L, magnesium sulfate heptahydrate at 12 g/L) was added while controlling dissolved oxygen.

[0189] (3) When the absorbance at 600 nm reached about 100, the preset temperature was controlled to be lowered to 25° C. After confirming that the temperature reached the thus set temperature, 1.1 mL of 0.5 mM IPTG was added, and culture was continued at 25° C. Culture was terminated about 42 hours after the start of culture, and the culture solution was centrifuged at 4° C. and 8000 rpm for 20 minutes, thereby collecting bacterial cells.

[0190] (4) The bacterial cells collected in (3) were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing 0.5M sodium chloride and 20 mM imidazole to yield a concentration of 5 mL/1 g (of cells), and then the cells were disrupted using an ultrasonic generator (Insonator 201M [manufactured by KUBOTA Corporation co., ltd.]) at 8° C. and an output power of about 150 W for about 10 minutes. A solution of the disrupted cells was centrifuged twice at 4° C. and 8000 rpm for 20 minutes, thereby collecting the supernatant.

[0191] (5) The supernatant obtained in (4) was applied to an XK26/20 column column (manufactured by Cytiva) filled with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) which was preliminarily equilibrated with Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride and 20 mM imidazole (hereinafter, also referred to as “equilibrium solution A”). After washing with the equilibration solution A, elution was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0192] (6) The eluate obtained in (5) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 4 mM imidazole and 150 mM sodium chloride, and then applied to an XK26/20 column column (manufactured by Cytiva) filled with 50 mL of TALON Metal affinity Resin (manufactured by Takara Bio Inc.) equilibrated with the same buffer solution. After washing with the buffer used for equilibration, elution was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0193] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, thereby preparing about 200 mg of AVR5eHC protein.

Example 11 Preparation of Viral Vector (AAV2-EGFP)

[0194] (1) A nucleotide sequence (SEQ ID NO: 52) was designed, in which a restriction enzyme EcoRI recognition sequence (GAATTC) was added to the 5′ end side of a polynucleotide encoding an enhanced green fluorescent protein (EGFP) consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3′ end side of the polynucleotide.

[0195] (2) The polynucleotide consisting of the sequence set forth in SEQ ID NO: 52 was totally synthesized and cloned into a plasmid (consigned to FASMAC, named pUC-EGFP). The E. coli JM109 strain was transformed with pUC-EGFP, and the obtained transformant was cultured. pUC-EGFP was extracted from the culture solution using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0196] (3) pUC-EGFP obtained in (2) was digested with restriction enzymes EcoRI and BamHI, and ligated to an expression vector pAAV-CMV (manufactured by Takara Bio Inc.), which was preliminarily digested with restriction enzymes EcoRI and BamHI. The E. coli JM109 strain was transformed using the ligation product. After the obtained transformant was cultured in LB medium containing 100 μg/mL carbenicillin, an expression vector pAAV-EGFP expressing EGFP was extracted using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0197] (4) The transformant obtained in (3) was cultured with shaking overnight at 37° C. in a 5 L baffled flask containing 1 L of 2YT medium supplemented with 100 μg/mL carbenicillin. After the end of culture, the cells were collected by centrifugation. A large amount of pAAV-EGFP was prepared from the recovered cells using a Plasmid Mega Kit (manufactured by QIAGEN).

[0198] (5) Preparation of AAV2-EGFP

[0199] (5-1) The E. coli JM109 strain was transformed using pRC2-mi342 Vector (manufactured by Takara Bio Inc.) and pHelper Vector (manufactured by Takara Bio Inc.). Large amounts of pRC2-mi342 and pHelper were prepared by performing the same operation as in (4) using the obtained transformant.

[0200] (5-2) HEK293T cells were cultured in ten T-225 flasks (manufactured by Thermo Fisher Scientific) containing 45 mL of D-MEM medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) containing 10% (v/v) bovine serum. Transfection was carried out by adding a complex of pAAV-EGFP prepared in (4), pRC2-mi342 and pHelper prepared in (5-1), and polyethyleneimine (manufactured by Polysciences Inc.) thereto, and the cells were statically cultured for 3 days under the conditions of 5% (v/v) carbon dioxide and 37° C. After the culture, the detached cells were recovered by centrifugation and each set of cells obtained from five T-225 flasks were cryopreserved at −80° C.

[0201] (5-3) The frozen cells obtained in (5-2) were thawed and suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 4 mM magnesium chloride, and 0.01% (w/v) Tween 20 (trade name). One-twentieth ( 1/20) the amount of Benzonase (manufactured by MERCK MILLIPORE) was added thereto, and the mixture was left to stand sill at 37° C. for 1 hour and then centrifuged at 13000×g at 4° C. for 10 minutes, thereby obtaining a supernatant. Ammonium sulfate was added to the obtained supernatant so as to result in 15% saturation, and the mixture was centrifuged again under the same conditions. The obtained supernatant was applied to a filter having a pore size of 0.45 μm, and suspended matter was removed.

[0202] (5-4) The supernatant from which suspended matter was removed was applied to a 5-mL AVB Sepharose column (manufactured by Cytiva) which was preliminarily equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter, also referred to as “equilibrium solution B”). After washing with the equilibration solution B, elution was performed with 0.1M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The obtained eluate was neutralized by adding one-fourth (¼) the amount of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride, thereby obtaining a solution of AAV2-EGFP which is an AAV vector. The AAV2-EGFP concentration in the solution was quantified by qPCR using an AAVpro Titration Kit (manufactured by Takara Bio Inc.).

Example 12 Preparation of AAV-Binding Protein-Immobilized Gel (Part 1)

[0203] (1) Gel into which a maleimide group was introduced was prepared by chemically modifying the hydroxy group on the surface of a hydrophilic vinyl polymer for a separating agent (manufactured by Tosoh Corporation: TOYOPEARL). To 3.5 g of the prepared gel, 28 mg of the AVR5eHC protein prepared in Example 10 and Tris(2-carboxyethyl)phosphine (TCEP) with a final concentration of 0.1 mM as a reducing agent were added. The reaction was carried out by shaking at pH 7.4 and 4° C. for 15 hours. As a result, gel on which AVR5e was immobilized (named AVR5e-immobilized gel) was prepared.

[0204] (2) A Tricorn 10/50 column (manufactured by Cytiva) was filled with 2 mL of the AVR5e-immobilized gel prepared in (1), thereby preparing a column (named AVR5e column). The AVR5e column was connected to AKTAprime plus (manufactured by Cytiva) and equilibrated with 50 mM phosphate buffer (pH 7.4) (hereinafter also referred to as “equilibrium solution C”) containing 150 mM sodium chloride and 0.05% (w/v) Tween 20 (trade name), and then 10 mL of the cell extract containing AAV2-EGFP obtained in Example 11 (5-3) was applied thereto at a flow rate of 1 mL/min.

[0205] (3) After washing with the equilibration solution C, AAV2-EGFP was eluted with 0.1M acetate buffer (pH 2.5) containing 0.5 M sodium chloride from the AVR5e column. The eluate was fractionated in 1 ml aliquots and collected as fractions. The recovered fractions were neutralized by adding 250 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0206] (4) SDS-PAGE and silver staining using a Pierce Silver Stain Kit (manufactured by Thermo Fisher Scientific) were carried out to compare the purity of the eluate obtained in (3) with the purity of the cell extract obtained in Example 10 (5-3), which was a sample before purification.

[0207] The chromatographic pattern for the AVR5e column obtained in (3) is shown in FIG. 2, and the results of SDS-PAGE in (4) are shown in FIG. 3. The peak of AAV2-EGFP appeared around an elution time of 20 minutes (FIG. 2). When fractions (Fr6 to Fr9) corresponding to the peak were subjected to SDS-PAGE, only the bands corresponding to VP1, VP2, and VP3 constituting AAV were confirmed (FIG. 3). On the other hand, innumerable bands derived from contaminants were confirmed for the cell extract applied to the AVR5e column, in addition to the above bands (FIG. 3). The above results clarify that AAV can be purified with high purity by an AAV adsorbent containing gel and the AAV-binding protein of the invention immobilized on the gel.

[0208] Example 13 Creation and Screening of Mutation Library of AAV-Binding Proteins (Part 3) Among the mutated AAV-binding proteins evaluated in Example 7, AVR5e (Tyr342Cys-Gly390Ser-Lys399Glu-Ser476Arg-Asn487Asp substitution product, SEQ ID NO: 46) was selected. The polynucleotide portion encoding the protein was subjected to random mutagenesis by error-prone PCR.

[0209] (1) Using the plasmid pET-AVR5e into which the polynucleotide (SEQ ID NO: 47) encoding AVR5e (SEQ ID NO: 46) was inserted as a template, a reaction solution having the composition listed in Table 6 was prepared, and then error-prone PCR was performed under the same temperature conditions as in Example 3 (1). Mutations were successfully introduced into the polynucleotide encoding AVR5e by the error-prone PCR, and the average mutation rate was 1.7 amino acid mutations per molecule.

[0210] (2) After purifying the PCR product obtained in (1), a random mutant library was prepared by the methods described in Examples 6 (2) and (3). The library (transformants) was cultured by the methods described in Example 3 (4) and (5).

[0211] (3) The culture solution of (2) was centrifuged, and the obtained culture supernatant was diluted 32-fold with ultrapure water. The diluted culture solution in an amount of 60 μL and 60 μL of 0.1 M glycine hydrochloric acid buffer (pH 3.0) were mixed, and heat treatment and acid treatment were performed at 61.0° C. for 15 minutes.

[0212] (4) The binding activity of AAV-binding protein to VLP2 when the treatment of (3) was performed and the binding activity of AAV-binding protein to VLP2 when the treatment of (3) was not performed were measured by the ELISA method described in Example 2 (6). The residual activity was calculated by dividing the binding activity of AAV-binding protein to VLP2 when the heat treatment was performed by the binding activity of AAV-binding protein to VLP2 when the heat treatment was not performed.

[0213] (5) A random mutant library of about 1800 transformants was evaluated by the method of (4), and among them, transformants expressing AAV-binding proteins having improved residual activity as compared with the parent molecule AVR5e were selected. Each selected transformant was cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0214] (6) The nucleotide sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and the amino acid mutation site was identified.

[0215] Table 13 summarizes the amino acid substitution positions of the AAV-binding proteins expressed by the transformants selected in (5) above with respect to AVR5e (Tyr342Cys-Gly390Ser-Lys399Glu-Ser476Arg-Asn487Asp substitution product) and the residual activity (%) thereof after heat treatment and acid treatment.

[0216] It can be said that an AAV-binding protein, in which amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 have at least one of the following amino acid substitutions, has improved heat stability and acid stability as compared with the wild-type AAV-binding protein: Val317Asp, Gln318Pro, Thr320Ala, Asn324Asp, Val326Ala, Val326Glu, Leu328Pro, Asn329His, Val332Ala, Lys362Glu, Lys362Asn, Lys371Glu, Lys371Asn, Leu376Pro, Glu378Gly, Glu378Val, Phe379Ser, Phe379Tyr, Val381Asp, Gly392Cys, Thr397Ser, Glu401Val, Arg406His, Asn492Asp, and Val499Glu.

TABLE-US-00013 TABLE 13 No. Amino acid substitution Residual activity [%] No. Amino acid substitution Residual activity [%] 85 Val317Asp 50.2 94 Val326Glu, Asn329His 44.9 86 Lys371Glu 41.6 95 Val326Ala, Lys362Glu 45.3 87 Lys371Asn 56.5 96 Val326Glu, Phe379Ser 60.9 88 Leu376Pro 41.7 97 Val332Ala, Lys371Glu 49.6 89 Glu378Gly 46.1 98 Leu376Pro, Asn492Asp 47.6 90 Gly392Cys 53.3 99 Gln318Pro, Phe379Tyr, Glu401Val 54.0 91 Val317Asp, Lys371Asn 55.7 100 Asn324Asp, Lys362Glu, Val499Glu 50.1 92 Thr320Ala, Val381Asp 52.4 101 Val326Glu, Asn329His, Glu378Val 48.5 93 Asn324Asp, Lys362Glu 50.7 102 Leu328Pro, Lys362Asn, Thr397Ser, Arg406His 41.1 AVR5e 25.5

Example 14 Integration of Stabilized Amino Acid Substitutions (Part 2)

[0217] By integrating the amino acid substitutions involved in the improvement of heat stability and acid stability of the AAV-binding proteins found in Example 13 in AVR5e (SEQ ID NO: 46), the stability was further improved. Specifically, among the amino acid-substituted (mutated) AAV-binding proteins listed in Table 13, No. 93 (integrated amino acid substitutions of Asn324Asp and Lys362Glu for AVR5e) (named AVR7a) and No. 91 (integrated amino acid substitutions of Val317Asp and Lys371Asn for AVR5e) (named AVR7b) were selected. The amino acid substitutions described in the following (a) or (b) were integrated into the proteins. The Asn324Asp and Lys362Glu substitutions in the amino acid sequence of AVR7a (SEQ ID NO: 53) are located at positions 37 and 75 in SEQ ID NO: 53, respectively. In addition, the Val317Asp and Lys371Asn substitutions in the amino acid sequence of AVR7b (SEQ ID NO: 55) are located at positions 30 and 84 in SEQ ID NO: 55, respectively.

(a) AVR7a with Val317Asp introduced therein (named AVR8a)
(b) AVR7b with Lys362Glu introduced therein (named AVR8g)

[0218] Hereinafter, a method for producing the following AAV-binding proteins will be described in detail.

[0219] (a) AVR8a

[0220] From the amino acid substitutions clarified in Example 13 that are involved in improving the stability to heat and acid, Val317Asp was selected and integrated in AVR7a (SEQ ID NO: 53), thereby preparing AVR8a. Specifically, AVR8a was prepared by introducing a mutation that yields Val317Asp in the polynucleotide (SEQ ID NO: 54) encoding AVR7a (SEQ ID NO: 53).

[0221] (a-1) A reaction solution having the composition listed in Table 12 was prepared using a plasmid pET-AVR7a into which the polynucleotide (SEQ ID NO: 54) encoding AVR7a was inserted as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 57 (5′-TTTT[GGTCTC]AGCAGGCGAGTCGGATCAGATTACCC-3′) and SEQ ID NO: 58 (5′-TTTT[GGTCTC]ACTGCAGAGCCCATGGCCATCGCCGG-3′) (both the square brackets in SEQ ID NO: 57 and the square brackets in SEQ ID NO: 58 indicate the restriction enzyme BsaI recognition sequence) as PCR primers. The reaction solution was heat-treated at 98° C. for 5 minutes, a reaction including a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, and a third step at 72° C. for 6 minutes was repeated 30 cycles, and lastly, heat treatment was performed at 72° C. for 5 minutes. Thus, PCR was performed.

[0222] (a-2) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN). The purified PCR product was named 8ap.

[0223] (a-3) The Bap obtained in (a-2) was treated with the restriction enzyme DpnI (manufactured by NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The thus obtained gene was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

[0224] (a-4) The DpnI-treated product obtained in (a-3) was digested with a restriction enzyme BsaI (manufactured by NEB) under the reaction solution having the composition listed in Table 10 and ligated with T4DNA ligase (manufactured by NEB).

[0225] (a-5) The E. coli BL21 (DE3) strain was transformed using the ligation product of (a-4). The obtained transformant was cultured in LB medium supplemented with 50 μg/mL kanamycin. Plasmids were extracted from the collected bacterial cells (transformants), thereby obtaining a plasmid pET-AVR8a containing a polynucleotide encoding AVR8a with eight amino acid substitutions in the wild-type AAV-binding protein.

[0226] (a-6) The nucleotide sequence of pET-AVR8a was analyzed in the same manner as in Example 1 (5).

[0227] The amino acid sequence of AVR8a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 59, and the sequence of the polynucleotide encoding AVR8a is set forth in SEQ ID NO: 60. In SEQ ID NO: 59, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR8a (domains 1 and 2 corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 59, aspartic acid of Val317Asp is present at position 30, aspartic acid of Asn324Asp is present at position 37, cysteine of Tyr342Cys is present at position 55, glutamic acid of Lys362Glu is present at position 75, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

[0228] (b) AVR8g

[0229] From the amino acid substitutions clarified in Example 13 that are involved in improving the stability to heat and acid, Lys362Glu was selected and accumulated in AVR7b (SEQ ID NO: 55), thereby preparing AVR8g. Specifically, AVR8g was prepared by introducing a mutation that yields Lys362Glu in the polynucleotide (SEQ ID NO: 56) encoding AVR7b (SEQ ID NO: 55).

[0230] (b-1) A plasmid pET-AVR8g containing the polynucleotide encoding AVR8g with eight amino acid substitutions in the wild-type AAV-binding protein was obtained in the same manner as in (a-1) to (a-5) except that a plasmid pET-AVR7b into which the polynucleotide (SEQ ID NO: 56) encoding AVR7b was inserted was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 61 (5′-TTTT[GGTCTC]ACGAGATGGAAGGAGAACATTCGCAG-3′) and SEQ ID NO: 62 (5′-TTTT[GGTCTC]ACTCGCCGCTGTAGTCGCGAGG-3′) (both the square brackets in SEQ ID NO: 61 and the square brackets in SEQ ID NO: 62 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0231] (b-2) The nucleotide sequence of pET-AVR8g was analyzed in the same manner as in Example 1 (5).

[0232] The amino acid sequence of AVR8g with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 63, and the sequence of the polynucleotide encoding AVR8g is set forth in SEQ ID NO: 64. In SEQ ID NO: 63, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR8g (domains 1 and 2 corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 63, aspartic acid of Val317Asp is present at position 30, cysteine of Tyr342Cys is present at position 55, glutamic acid of Lys362Glu is present at position 75, aspartic acid of Lys371Asn is present at position 84, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

Example 15 Evaluation of Acid Stability of Mutated AAV-Binding Protein (Part 3)

[0233] (1) The transformants expressing the mutated AAV-binding protein AVR7a obtained in Example 13 and the mutated AAV-binding proteins (AVR8a and AVR8g) prepared in Example 14 were each cultured by the methods described in Examples 3 (4) and (5), and then the culture solutions were centrifuged, thereby obtaining culture supernatants containing the expressed mutated AAV-binding proteins.

[0234] (2) The binding activity of the mutated AAV-binding protein in each culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, each culture supernatant obtained in (1) was diluted with pure water such that the measured values would be the same.

[0235] (3) Each diluted mutated AAV-binding protein solution was divided into three portions, and each portion was mixed with an equal amount of 0.1 M glycine-HCl buffer (pH 3.0). Two of the three samples were heat-treated at 80.8° C. and 89.8° C., respectively, for 15 minutes, and the remaining one was left to stand still at room temperature (25° C.) for 15 minutes. Thereafter, the pH was adjusted to around 6 by mixing each sample with 0.5 M MES buffer (pH 6.0) at a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6).

[0236] (4) The residual activity was calculated by dividing the absorbance at 450 nm when the heat treatment and acid treatment described in (3) were performed by the absorbance at 450 nm when standing still was performed at room temperature.

[0237] Table 14 shows the results. Each of the mutated AAV-binding proteins (AVR8a and AVR8g) prepared in Example 14 had a residual activity higher than that of AVR7a (No. 93 in Table 14) when treated at 80.8° C. This clarifies that the two mutated AAV-binding proteins have significantly improved stability to heat treatment under acidic conditions, i.e., stability to acid and heat, as compared with the wild-type AAV-binding protein.

TABLE-US-00014 TABLE 14 Residual activity after AAV-binding protein heat treatment [%] SEQ ID NO Name Amino acid substitution 80.8° C. 89.8° C. 59 AVR8a Val317Asp, Asn324Asp, Tyr342Cys, Lys362Glu, 66 52 Gly390Ser, Lys399Glu, Ser476Arg, Asn487Asp 63 AVR8g Val317Asp, Tyr342Cys, Lys362Glu, Lys371Asn, 67 55 Gly390Ser, Lys399Glu, Ser476Arg, Asn487Asp 53 AVR7a Asn324Asp, Tyr342Cys, Lys362Glu, Gly390Ser, 57 54 Lys399Glu, Ser476Arg, Asn487Asp

Example 16 Creation and Screening of Mutation Library of AAV-Binding Proteins (Part 4)

[0238] Among the mutated AAV-binding proteins evaluated in Example 15, AVR8g (Val317Asp-Tyr342Cys-Lys362Glu-Lys371Asn-Gly390Ser-Lys399Glu-Ser476Arg-Asn487Asp substitution product, SEQ ID NO: 63) was selected. The polynucleotide portion encoding the protein was subjected to random mutagenesis by error-prone PCR.

[0239] (1) Using the plasmid pET-AVR8g into which the polynucleotide (SEQ ID NO: 64) encoding AVR8g (SEQ ID NO: 63) was inserted as a template, a reaction solution having the composition listed in Table 6 was prepared, and then error-prone PCR was performed under the same temperature conditions as in Example 3 (1). Mutations were successfully introduced into the polynucleotide encoding AVR8g by the error-prone PCR, and the average mutation rate was 2.3 amino acid mutations per molecule.

[0240] (2) After purifying the PCR product obtained in (1), a random mutant library was prepared by the methods described in Examples 6 (2) and (3). The library (transformants) was cultured by the methods described in Example 3 (4) and (5).

[0241] (3) The culture solution of (2) was centrifuged, and the obtained culture supernatant was diluted 16-fold with ultrapure water. The diluted culture solution in an amount of 60 μL and 60 μL of 0.1 M glycine-HCl buffer (pH 10.0) were mixed, and heat treatment and acid treatment were performed at 41.2° C. for 15 minutes.

[0242] (4) The binding activity of AAV-binding protein to VLP2 when the treatment of (3) was performed and the binding activity of AAV-binding protein to VLP2 when the treatment of (3) was not performed were measured by the ELISA method described in Example 2 (6). The residual activity was calculated by dividing the binding activity of AAV-binding protein to VLP2 when the heat treatment was performed by the binding activity of AAV-binding protein to VLP2 when the heat treatment was not performed.

[0243] (5) A random mutant library of about 1800 transformants was evaluated by the method of (4), and among them, transformants expressing AAV-binding proteins having improved residual activity as compared with the parent molecule AVR8g were selected. Each selected transformant was cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (manufactured by QIAGEN).

[0244] (6) The nucleotide sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and the amino acid mutation site was identified.

[0245] (7) Table 15 summarizes the amino acid substitution positions of the AAV-binding proteins expressed by the transformants selected in (6) above with respect to AVR8g (Val317Asp-Tyr342Cys-Lys362Glu-Lys371Asn-Gly390Ser-Lys399Glu-Ser476Arg-Asn487Asp substitution product) and the residual activity (%) thereof after heat treatment and acid treatment.

[0246] It can be said that an AAV-binding protein, in which amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 have at least one of the following amino acid substitutions, has improved heat stability and alkali stability as compared with the wild-type AAV-binding protein: Ser312Pro, Ala313Ser, Lys323Arg, Asn324Ser, Gln327His, Gln327Leu, Asn329Ile, Glu335Gly, Glu335Val, Glu340Val, Thr341Pro, Thr343Ser, Tyr344Phe, Trp346Cys, Arg353Cys, Tyr355Cys, Ser356Cys, Gly357Cys, Met359Leu, Glu360Lys, Glu360Val, Gly361Cys, Lys(Glu)362Gly, Ser364Leu, Ile366Thr, Leu369Gln, Lys(Asn)371Asp, Leu372Pro, Leu372Gln, Thr373Ala, Pro374Leu, Leu376Pro, Glu378Gly, Glu378Val, Phe379Cys, Phe379Ser, Lys380Glu, Val381Ala, Ile382Val, Val383Ala, Glu384Val, Gln386Arg, His389Arg, Tyr393Cys, Val394Asp, Val394Ile, Asn395Ser, Val396Ala, Arg403His, Gln415Arg, Thr426Ala, Gln432Leu, Gln432Arg, Gln441Arg, His443Leu, Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, and Val499Ile.

TABLE-US-00015 TABLE 15 No. Amino acid substitution Residual activity [%] No. Amino acid substitution Residual activity [%] 103 Glu340Val 35.0 128 Ser312Pro, Pro374Leu 74.9 104 Tyr344Phe 40.3 129 Ala313Ser, Glu378Gly 67.5 105 Arg353Cys 62.1 130 Lys323Arg, Glu384Val 63.1 106 Tyr355Cys 63.8. 131 Asn324Ser, Gly357Cys 70.3 107 Ser356Cys 67.6 132 Gln327His, Glu378Val 56.5 108 Met359Leu 32.4 133 Gln327Leu, Lys(Asn)371Asp 62.1 109 Glu360Lys 40.2 134 Asn329Ile, Val499Ile 63.9 110 Glu360Val 33.3 135 Glu335Gly, Gln386Arg 36.3 111 Lys(Glu)362Gly 41.1 136 Glu335Val, Thr343Ser 38.8 112 Ser364Leu 45.3 137 Thr341Pro, Phe379Ser 86.3 113 Ile366Thr 41.4 138 Gly361Cys, Gly(Ser)390Gly 51.5 114 Leu372Pro 91.6 139 Leu369Gln, Pro374Leu 85.2 115 Leu372Gln 72.8 140 Leu372Pro, Val396Ala 92.8 116 Thr373Ala 57.8 141 Leu376Pro, Gly(Ser)390Gly 85.0 117 Leu376Pro 76.7 142 Glu378Gly, Arg403His 76.6 118 Glu378Gly 66.7 143 Val381Ala, Ile382Val 90.3 119 Phe379Ser 24.3 144 Val383Ala, Gln415Arg 65.7 120 Lys380Glu 59.5 145 Trp346Cys, Arg353Cys, His389Arg 63.5 121 Val381Ala 89.9 146 Leu372Gln, Leu376Pro, Val394Asp 81.4 122 Gln386Arg 34.9 147 Phe379Cys, Gln432Leu, Ser488Leu 79.7 123 Tyr393Cys, 60.5 148 Glu384Val, Gln432Arg, Ile456Val 37.4 124 Gln441Arg 25.2 149 Val394Ile, Asn395Ser, Thr426Ala 70.5 125 His443Leu 36.5 150 Val396Ala, Gln441Arg, Lys448Glu 49.7 126 Lys448Glu 34.5 63 AVR8G 23.1 127 Asp483Asn 23.3

Example 17 Monomerization of Mutated AAV-Binding Protein (Part 1)

[0247] Among the mutated AAV-binding proteins found to have improved heat stability and alkali stability in Example 16, No. 143 (integrated amino acid substitutions of Val381Ala and Ile382Val for AVR8g) (named AVR10) was selected. An amino acid substitution of Tyr(Cys)342Ser was introduced therein to monomerize the protein (named AVR10s). Specifically, AVR10s was prepared by introducing a mutation that yields Tyr(Cys)342Ser in the polynucleotide (SEQ ID NO: 66) encoding AVR10 (SEQ ID NO: 65). The Val381Ala and Ile382Val substitutions in the amino acid sequence of AVR10 (SEQ ID NO: 65) are located at positions 94 and 95 in SEQ ID NO: 65, respectively.

[0248] (1) A plasmid pET-AVR10s containing the polynucleotide encoding AVR10s with ten amino acid substitutions in the wild-type AAV-binding protein was obtained in the same manner as in Example 14 (a-1) to (a-5) except that pET26b-AVR10 into which the polynucleotide (SEQ ID NO: 66) encoding AVR10 was inserted was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 67 (5′-TTTT[GGTCTC]AGAAACCTCTACGTATGACTG-3′) and SEQ ID NO: 68 (5′-TTTT[GGTCTC]ATTTCCCCTTTCGGTGG-3′) (both the square brackets in SEQ ID NO: 67 and the square brackets in SEQ ID NO: 68 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0249] (2) The nucleotide sequence of pET-AVR10s was analyzed in the same manner as in Example 1 (5).

[0250] The amino acid sequence of AVR10s with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 69, and the sequence of the polynucleotide encoding AVR10s is set forth in SEQ ID NO: 70. In SEQ ID NO: 69, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR10s (corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219. In SEQ ID NO: 69, aspartic acid of Val317Asp is present at position 30, serine of Tyr(Cys)342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

Example 18 Monomerization of Mutated AAV-Binding Protein (Part 2)

[0251] A mutated AAV binding protein was prepared, into which an amino acid substitution of Tyr(Cys)342Met was introduced to monomerize the mutated AAV binding protein AAV5eHC prepared in Example 9, and to which PKD3 (amino acid residues ranging from tyrosine at position 501 to lysine at position 597 in SEQ ID NO: 1) was further added (named AVR5eMHCD3). Specifically, AVR5eMHCD3 was prepared by introducing a mutation that yields Tyr(Cys)342Met in the polynucleotide (SEQ ID NO: 49) encoding AVR5eHC (SEQ ID NO: 50) and then adding PKD3 thereto.

[0252] (1) A plasmid pET-AVR5eMHC containing the polynucleotide encoding the mutated AAV-binding protein in which an amino acid substitution of Tyr(Cys)342Met was introduced into AVR5eHC was obtained in the same manner as in Example 14 (a-1) to (a-5) except that pET26b-AVR5eHC was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 71 (5′-TTTT[GGTCTC]AGAAACCATGACGTATGACTG-3′) and SEQ ID NO: 68 (both the square brackets in SEQ ID NO: 71 and the square brackets in SEQ ID NO: 68 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0253] (2) PCR was performed in the same manner as in Example 14 (a-1) except that pUC-AAVR was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 72 (5′-TTTT[GGTCTC]AGATGTTTGTTATTTTCGGGCTGAACAATC-3′) and SEQ ID NO: 73 (5′-TTTT[GGTCTC]AAGACTATCCGCCGGTAGCGAACGC-3′) (both the square brackets in SEQ ID NO: 72 and the square brackets in SEQ ID NO: 73 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0254] (3) PCR was performed in the same manner as in Example 14 (a-1) except that pET26b-AVR5eMHC prepared in (1) was used as a template and oligonucleotides consisting respectively of the sequences set forth in SEQ ID NO: 74 (5′-TTTT[GGTCTC]AGTCTACCGCTTTGTTG-3′) and SEQ ID NO: 75 (5′-TTTT[GGTCTC]ACATCATCATCATCATCAT-3′) (both the square brackets in SEQ ID NO: 74 and the square brackets in SEQ ID NO: 75 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0255] (4) The PCR products amplified in (2) and (3) were each subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN). The purified PCR product of (2) was named D3p, and the PCR product of (3) was named 5MVp.

[0256] (5) D3p and 5Mvp obtained in (4) were treated with the restriction enzyme DpnI (manufactured by NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The thus obtained genes were each subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

[0257] (6) The DpnI-treated product obtained in (5) was digested with a restriction enzyme BsaI (manufactured by NEB) under the reaction solution having the composition listed in Table 16 and ligated with T4DNA ligase (manufactured by NEB).

TABLE-US-00016 TABLE 16 Composition Volume 20 ng/μL PCR product (D3p) 5 μL 20 ng/μL PCR product (5MVp) 5 μL 10 × CutSmart buffer (manufactured by NEB) 1.5 μL BsaI (manufactured by NEB) 1 μL T4 DHA ligase buffer (manufactured by NEB) 1.5 μL T4 DHA ligase (manufactured by NEB) 1 μL H.sub.2O up to 15 μL

[0258] (7) The E. coli BL21 (DE3) strain was transformed using the ligation product of (6). The obtained transformant was cultured in LB medium supplemented with 50 μg/mL kanamycin. Plasmids were extracted from the collected bacterial cells (transformants), thereby obtaining a plasmid pET-AVR5eMHCD3 containing the polynucleotide encoding the mutated AAV-binding protein in which AVR5eHD was monomerized and to which PKD3 was further added.

[0259] (8) The nucleotide sequence of pET-AVR5eMHCD3 was analyzed in the same manner as in Example 1 (5).

[0260] The amino acid sequence of AVR5eMHCD3 is set forth in SEQ ID NO: 76, and the polynucleotide sequence thereof is set forth in SEQ ID NO: 77. In SEQ ID NO: 76, the PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV-binding protein AVR5eMHC (corresponding to the domain ranging from positions 312 to 500 of SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, the AAV extracellular domain 3 (PKD3) (the domain ranging from positions 501 to 597 of SEQ ID NO: 1) ranges from tyrosine (Tyr) at position 214 to lysine (Lys) at position 310, the histidine tag ranges from an amino acid at position 311 to histidine (His) at position 316, and the cysteine tag sequence as a tag for immobilization ranges from cysteine at position 317 to glycine (Gly) at position 323. In SEQ ID NO: 76, methionine of Tyr(Cys)342Met is present at position 55, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200.

Example 19 Preparation of Monomerized Mutated AAV-Binding Protein (Part 1)

[0261] (1) A transformant capable of expressing AVR10s obtained by transforming the E. coli BL21 (DE3) strain with pET-AVR10s prepared in Example 17 or a transformant capable of expressing AVR5eMHCD3 obtained by transforming the E. coli BL21 (DE3) strain with pET-AVR5eMHCD3 prepared in Example 18 were each inoculated in a 3 mL of 2YT liquid medium supplemented with 50 μg/mL kanamycin and aerobically cultured with shaking overnight at 37° C., thereby performing preculture.

[0262] (2) The preculture solution of (1) in a volume of 2 mL was inoculated in 200 mL of 2YT liquid medium supplemented with 50 μg/mL kanamycin in a 1 L baffled flask and aerobically cultured with shaking at 37° C.

[0263] (3) The flask was cooled on ice 2.0 hours after the start of culture, IPTG was added to yield a final concentration of 0.1 mM, and shaking culture was continued aerobically overnight at 25° C.

[0264] (4) After the end of culture, the cells were recovered by centrifuging the culture solution at 4° C. and 8000 rpm for 20 minutes.

[0265] (5) The bacterial cells collected in (4) were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole to yield a concentration of 5 mL/1 g (of cells), and then the cells were disrupted using an ultrasonic generator (Insonator 201M [manufactured by KUBOTA Corporation co., ltd.]) at 8° C. and an output power of about 150 W for about 10 minutes. A solution of the disrupted cells was centrifuged twice at 4° C. and 8000 rpm for 20 minutes, thereby collecting the supernatant.

[0266] (6) The supernatant obtained in (5) was applied to an XK26/20 column column (manufactured by Cytiva) filled with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) which was preliminarily equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter, also referred to as “equilibrium solution D”). After washing with the equilibration solution D, elution was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0267] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, thereby preparing AVR10s protein or AVR5eMHCD3 protein in the amount required for heat stability evaluation.

Example 20 Evaluation of Heat Stability of Mutated AAV-Binding Protein (Part 4)

[0268] (1) The binding activity of AVR10s prepared in Example 18 to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, the AVR10s solution was diluted with pure water such that the measured values would be the same.

[0269] (2) The diluted AVR10s solution was divided into four portions, each portion was mixed with an equal amount of 0.1 M glycine-HCl buffer (pH 3.0). Three of the four samples were heat-treated at 31.7° C., 41.2° C., and 50.0° C., respectively, for 15 minutes, and the remaining one was left to stand still at room temperature (25° C.) for 15 minutes. Thereafter, the pH was adjusted to around 6 by mixing each sample with 0.5 M MES buffer (pH 6.0) at a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6).

[0270] (3) The residual activity was calculated by dividing the absorbance at 450 nm when the heat treatment described in (2) was performed by the absorbance at 450 nm when standing still was performed at room temperature.

[0271] (4) As a comparison target, the heat stability of the wild-type AAV-binding protein (SEQ ID NO: 7) was evaluated in the same manner as in (1) to (3).

[0272] Table 17 shows the results. The mutated AAV-binding protein AVR10s prepared in Example 17 had a residual activity higher than that of the wild-type AAV-binding protein regardless of the treatment temperature. This clarifies that AVR10s has significantly improved stability to heat treatment under acidic conditions, i.e., stability to acid and heat, as compared with the wild-type AAV-binding protein.

TABLE-US-00017 TABLE 17 Residual activity after AAV-binding protein heat treatment [%] SEQ ID NO Name Amino acid substitution 31.7° C. 41.2° C. 50.0° C. 69 AVR10s Val317Asp, Tyr(Cys)342Ser, Lys362Glu, Lys371Asn, Val381Ala, 99 79 67 Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, Asn487Asp 7 Wild type 43 35 30

Example 21 Evaluation of Heat Stability of Mutated AAV-Binding Protein (Part 5)

[0273] (1) The binding activity of AVR5eMHCD3 prepared in Example 18 to VLP2 was measured using the ELISA method described in Example 2 (6). Based on the absorption at 450 nm corresponding to the above measurement results, AVR5eMHCD3 was diluted with pure water such that the measured values would be the same.

[0274] (2) The diluted AVR5eMHCD3 solution was divided into four portions, and each portion was diluted 2-fold with pure water. Three of the four samples were heat-treated at 51.7° C., 66.4° C., and 70.0° C., respectively, for 15 minutes, and the remaining one was left to stand still at room temperature (25° C.) for 15 minutes. Thereafter, the sample was mixed with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride in a ratio of 3:2, and the binding activity of the protein to VLP2 was measured by the ELISA method described in Example 2 (6).

[0275] (3) The residual activity was calculated by dividing the absorbance at 450 nm when the heat treatment described in (2) was performed by the absorbance at 450 nm when standing still was performed at room temperature.

[0276] (4) As a comparison target, the heat stability of the wild-type AAV-binding protein (SEQ ID NO: 7) was evaluated in the same manner as in (1) to (3).

[0277] Table 18 shows the results. The mutated AAV-binding protein AVR5eMHCD3 prepared in Example 18 had a residual activity higher than that of the wild-type AAV-binding protein regardless of the treatment temperature. This clarifies that AVR5eMHCD3 has significantly improved stability to heat, as compared with the wild-type AAV-binding protein.

TABLE-US-00018 TABLE 18 Residual activity after AAV-binding protein heat treatment [%] SEQ ID NO Name Amino acid substitution 31.7° C. 41.2° C. 50.0° C. 76 AVR5eMHCD3 Tyr(Cys)342Met, Gly390Ser, Lys399Glu, Ser476Arg, Asn487Asp 96 66 54 7 Wild type 72 41 34

Example 22 Preparation of AAV-Binding Protein-Immobilized Gel (Part 2)

[0278] (1) Wild-type AAV-binding protein-immobilized gel and AVR5eHC-immobilized gel were prepared in the same manner as in Example 12. Specifically, to 1 mL gel into which a maleimide group was introduced, 10 mg of AVR5eHC prepared in Example 10 or the wild-type AAV-binding protein and TCEP with a final concentration of 0.1 mM as a reducing agent were added. The reaction was carried out by shaking at pH 7.4 and 4° C. for 15 hours.

[0279] (2) The amount of each protein immobilized on gel was estimated by measuring the filtrate after immobilization in (1) with an absorptiometer and calculating the amount of non-immobilized protein. AVR5eHC was immobilized at 9 mg/mL (gel) on the AVR5eHC-immobilized gel, and the wild-type AAV-binding protein was immobilized at 10 mg/mL (gel) on the wild-type AAV-binding protein-immobilized gel.

[0280] (3) Each immobilized gel prepared in (1) was placed in an amount of 0.065 mL in Cosmo Spin Filter G (manufactured by Nacalai Tesque), thereby preparing a mini column (named AVR5eHC-immobilized gel mini column and wild-type AAV-binding protein-immobilized gel mini column, respectively). Each mini column was equilibrated with 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05% (w/v) Tween 20 (trade name) (hereinafter also referred to as “solution A”), and 2.9×10.sup.10 VG (number of vector genomes) of the AAV2-EGFP purified product obtained in Example 11 was applied thereto.

[0281] (4) Shaking was performed at 1600 rpm for 5 minutes using an incubator shaker MBR-022UP (manufactured by TAITEC CORPORATION), and then the filtrate was collected by centrifugation at 1000 G for 2 minutes. The solution A was added in an amount of 200 μL to each mini column again, and the mixture was shaken for 1 minute and then centrifuged to recover 400 μL together with the above filtrate (hereinafter, also referred to as “flow through”).

[0282] (5) Each column was washed two times with 400 μL of the solution A, and each filtrate was collected (the filtrates collected in the first and second times are also referred to as “wash 1” and “wash 2,” respectively). To each mini column, 200 μL of 50 mM phosphate buffer (pH 2.5) containing 150 mM sodium chloride and 0.05% (w/v) Tween 20 (trade name) (hereinafter, also referred to as “solution B”) was added. Shaking was performed for 5 minutes, and then the eluate was collected by centrifugation. The solution B was added in an amount of 200 μL to each mini column again, and the mixture was shaken for 1 minute and then centrifuged to recover 400 μL together with the above eluate (hereinafter, also referred to as “eluate 1”).

[0283] (6) Further, 400 μL of the solution B was added, and the mixture was centrifuged to collect the eluate (hereinafter, also referred to as “eluate 2”). The eluates 1 and 2 were neutralized by adding 100 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0284] (7) The numbers of AAV2-EGFP vector genomes contained in the flow through obtained in (3), wash 1, wash 2, and eluate 1 obtained in (5), and eluate 2 obtained in (6) were quantified by qPCR using AAVpro Titration Kits (manufactured by Takara Bio Inc.).

[0285] FIG. 4 shows the results. In the wild-type AAV-binding protein-immobilized gel mini column, most of the applied AAV was confirmed in the flow through fraction. Meanwhile, in the AVR5eHC-immobilized gel mini column, almost all of the applied AAV was adsorbed on the gel, and elution from the gel was confirmed in the eluate 1 fraction. The above results clarify that

[0286] AAV can be purified with higher efficiency by an AAV adsorbent containing gel and the AAV-binding protein of the invention immobilized on the gel than when wild-type AVR is used.

Example 23 Preparation of AAV-Binding Protein-Immobilized Gel (Part 3)

[0287] (1) By the same method as in Example 9, pET-AVR3 and pET-AVR8g were used as templates, thereby obtaining pET-AVR3HC and pET-AVR8gHC. Further, AVR3HC (protein having an immobilization tag at the C-terminus of AVR3) and AVR8gHC (protein having an immobilization tag at the C-terminus of AVR8g) were prepared in the same manner as in Example 10. AVR3HC-immobilized gel and AVR8gHC-immobilized gel were prepared using the prepared proteins in the manner as in Example 22. In addition, wild-type AAV-binding protein-immobilized gel was prepared in the same manner as in Example 22.

[0288] (2) The amount of each protein immobilized on gel was estimated by measuring the filtrate after immobilization in (1) with an absorptiometer and calculating the amount of non-immobilized protein. AVR3HC was immobilized at 3.8 mg/mL (gel) on the AVR3HC-immobilized gel, and AVR8gHC was immobilized at 5.7 mg/mL (gel) on the AVR8gHC-immobilized gel.

[0289] (3) Cosmo Spin Filter G (manufactured by Nacalai Tesque) was filled with 0.065 mL of the wild-type AAV-binding protein-immobilized gel, 0.065 mL of the AVR3HC-immobilized gel, and 0.018 mL of the AVR8gHC-immobilized gel prepared in (1), respectively, thereby preparing mini columns (named wild-type AAV-binding protein-immobilized gel mini column, AVR3HC-immobilized gel mini column, and AVR8gHC-immobilized gel mini column, respectively). Each mini column was equilibrated with 50 mM acetate buffer (pH 6.0) containing 150 mM sodium chloride (hereinafter also referred to as “solution A”). Subsequently, the AAV2-EGFP purified product obtained in Example 11 was applied to the flow through fraction in a plurality of times until it was confirmed that AAV2-EGFP was sufficiently leaked. Eventually, the amount of applied AAV2-EGFP per column was 2.9×10.sup.10 VG for the wild-type AAV-binding protein-immobilized gel mini column, 3.9×10.sup.10 VG for the AVR3HC-immobilized gel mini column, and 2.4×10.sup.11 VG for the AVR8gHC-immobilized gel mini column.

[0290] (4) To elute and recover AAV2-EGFP bound to the gel, each column was washed two times with 200 μL of the solution A, and each filtrate was recovered (hereinafter, also referred to as “wash 1”). To each mini column, 200 μL of 50 mM phosphate buffer (pH 3.0) containing 150 mM sodium chloride (hereinafter, also referred to as “solution B”) was added. Shaking was performed for 5 minutes, and then the eluate was collected by centrifugation. The solution B was added in an amount of 200 μL to each mini column again, and the mixture was shaken for 1 minute and then centrifuged to recover 400 μL together with the above eluate (hereinafter, also referred to as “eluate 1”).

[0291] (5) Further, 400 μL of the solution B was added, and the mixture was centrifuged to collect the eluate (hereinafter, also referred to as “eluate 2”). The eluates 1 and 2 were neutralized by adding 100 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0292] (6) The numbers of AAV2-EGFP vector genomes contained in the flow through, wash 1, eluate 1, and eluate 2 obtained in (3) to (5) were quantified by qPCR using AAVpro Titration Kits (manufactured by Takara Bio Inc.).

[0293] FIG. 5 shows the results. It was found that most of the AAV2-EGFP bound to the eluate1 fraction was eluted and recovered. From the amount of AAV2-EGFP recovered by elution, the amount of AAV2-EGFP bound to each mini column was converted per 1 mL of protein-immobilized gel. As a result, it was 8.6×10.sup.9 VG/mL (gel) for wild-type AAV-binding protein-immobilized gel, 2.8×10.sup.12 VG/mL (gel) for AVR3HC-immobilized gel, and 1.2×10.sup.13 VG/mL (gel) for AVR8gHC-immobilized gel.