Phage Display Vectors and Methods of Use

Abstract

The present invention relates to vectors suitable for use in displaying proteins on the surface of bacteriophage M13 as fusion constructs with the surface protein P.III, bacteriophage M13 particles comprising a mutated P.III protein on the phage coat surface, as well as methods for producing bacteriophage M13 particles and methods for transfecting or infecting a host cell comprising the vectors and bacteriophage of the invention.

Claims

1. A type 33 bacteriophage M13 vector comprising a first polynucleotide sequence encoding a polypeptide sequence as given by SEQ ID NO:1 and a second polynucleotide sequence encoding a polypeptide sequence as given by SEQ ID NO:2.

2. The type 33 bacteriophage M13 vector according to claim 1 wherein said first polynucleotide sequence is given by SEQ ID NO:3 and said second polynucleotide sequence is given by SEQ ID NO:4.

3. The type 33 bacteriophage M13 vector according to claim 1 further comprising a polynucleotide sequence encoding a suitable detection tag sequence cloned in-frame with and upstream of the first polynucleotide sequence encoding the polypeptide sequence as given by SEQ ID NO:1.

4. The type 33 bacteriophage M13 vector according to claim 1 further comprising a polynucleotide sequence encoding a suitable detection tag sequence cloned in-frame with and upstream of the first polynucleotide sequence encoding the polypeptide sequence as given by SEQ ID NO:1, wherein the polynucleotide sequence encoding the tag sequence encodes a c-myc tag, HA-tag, His-tag, Flag-tag, or a S-tag.

5. The type 33 bacteriophage M13 vector according to claim 4, wherein the polynucleotide sequence encoding the tag sequence encodes a c-myc tag.

6. The type 33 bacteriophage M13 vector according to claim 5 wherein the polynucleotide sequence encoding the c-myc tag is given by SEQ ID NO:8.

7. The type 33 bacteriophage M13 vector according to claim 1 wherein said vector comprises the polynucleotide sequence as given by SEQ ID NO:15.

8. The type 33 bacteriophage M13 vector according to claim 3 further comprising a polynucleotide sequence encoding an exogenous polypeptide cloned in-frame with and upstream of the polynucleotide sequence encoding the polypeptide sequence as given by SEQ ID NO:1, or upstream of the polynucleotide sequence encoding the suitable detection tag sequence.

9. The type 33 bacteriophage M13 vector according to claim 1 wherein said vector is a double stranded DNA molecule.

10. The type 33 bacteriophage M13 vector according to claim 1 wherein said vector is a double stranded DNA plasmid.

11. A method for producing a bacteriophage M13 particle comprising: (a) transfecting a bacterial host cell with a double stranded type 33 bacteriophage M13 vector comprising a first polynucleotide sequence encoding a polypeptide sequence as given by SEQ ID NO:1 and a second polynucleotide sequence encoding a polypeptide sequence as given by SEQ ID NO:2, (b) incubating said bacterial host cell under conditions suitable for expression of said first and second polynucleotide sequences and assembly of bacteriophage M13 particles in said bacterial host cell, and (c) recovering from said bacterial host cell a bacteriophage M13 particle comprising polypeptide sequences given by the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 independently displayed on the bacteriophage M13 coat surface.

12. The method according to claim 11 wherein said first polynucleotide sequence is given by SEQ ID NO:3 and said second polynucleotide sequence is given by SEQ ID NO:4.

13. The method according to claim 11 wherein the double stranded bacteriophage M13 vector further comprises a polynucleotide sequence encoding a suitable detection tag sequence cloned in-frame with and upstream of the first polynucleotide sequence encoding the polypeptide sequence as given by SEQ ID NO:1.

14. The method according to claim 13, wherein the polynucleotide sequence encoding the tag sequence encodes a c-myc tag, HA-tag, His-tag, Flag-tag, or and S-tag.

15. The method according to claim 13, wherein the polynucleotide sequence encoding the tag sequence encodes a c-myc tag.

16. The method according to claim 15 wherein the polynucleotide sequence encoding the c-myc tag is given by SEQ ID NO:8.

17. The method according to claim 11 wherein the double stranded M13 vector comprises the polynucleotide sequence as given by SEQ ID NO:15.

18. The method according to claim 11 wherein the double stranded M13 vector further comprises a polynucleotide sequence encoding an exogenous polypeptide cloned in-frame with and upstream of the polynucleotide sequence encoding the polypeptide sequence given by SEQ ID NO:1, or upstream of the polynucleotide sequence encoding the suitable detection tag sequence.

19. The method according to claim 11 wherein the bacterial host cell is an F.sup.+ bacterial strain.

20. A bacteriophage M13 particle wherein said particle comprises the polypeptides given by the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 independently displayed on the phage particle coat surface.

21. The bacteriophage M13 particle according to claim 20 wherein said particle further comprises a suitable detection tag sequence fused to the N-terminus of the polypeptide sequence given by SEQ ID NO:1.

22. The bacteriophage M13 particle according to claim 21 wherein the suitable detection tag sequence is given by SEQ ID NO:7.

23. The bacteriophage M13 particle according to claim 21 wherein said particle further comprises an exogenous polypeptide fused to the N-terminus of the polypeptide sequence given by the amino acid sequence of SEQ ID NO:1, or to the N-terminus of the suitable detection tag sequence.

24. The bacteriophage M13 particle according to claim 23 wherein the exogenous polypeptide is fused to the polypeptide sequence given by SEQ ID NO:1, or to the suitable detection tag sequence, via a peptide linker fused to the N-terminus of the polypeptide sequence given by SEQ ID NO:1, or to the N-terminus of the suitable detection tag sequence, and the C-terminus of the exogenous polypeptide.

Description

EXAMPLE 1

Display of c-myc tag-P.III Fusion on Bacteriophage M13 Surface

[0027] Using a type-33 bacteriophage M13 parent vector comprising both a wild-type g.III gene (SEQ ID NO:5) and a recombinant g.III gene (SEQ ID NO:3) under control of a lacZ promoter, each gene encoding a copy of the P.III surface protein and using nucleotide sequences encoding the endogenous P.III and pelB signal peptides, respectively (SEQ ID NOs:17 and 19), single amino acids in the N1 region of the wild-type g.III are randomized essentially as described above. Modified vectors comprising mutations which encode L8P or S11P substitutions in the mature wild-type g.III gene product are constructed and transfected into E.Coli l XL1-Blue cells to express a c-myc tag-encoding sequence (SEQ ID NO:8) cloned in-frame with and upstream of the recombinant g.III gene. The resulting P.III-c-myc fusion protein is displayed on the surface of harvested M13 particles and c-myc display levels are detected by a phage-titer dependent ELISA essentially as described above.

[0028] Table 1, below, provides the nucleic acid sequences of the parental and mutated wild-type g.III genes, the recombinant g.III gene, the c-myc tag-encoding sequence, the signal peptide-encoding sequences and the resulting amino acid sequences encoded thereby. Table 2 provides the results of the phage-titer dependent ELISA.

TABLE-US-00001 TABLE 1 Nucleic acid sequences Mutated vector Mutated vector (wild-type g.III (wild-type g.III Parental gene encoding gene encoding Vector vector, (wild L8P S11P component type g.III gene) substitution) substitution) WT g.III gene SEQ ID NO: 5 SEQ ID NO: 23 SEQ ID NO: 24 Recombinant SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 3 g.III gene c-myc tag gene SEQ ID NO: 8 SEQ ID NO: 8 SEQ ID NO: 8 WT g.III SEQ ID NO: 17 SEQ ID NO: 17 SEQ ID NO: 17 signal sequence Recombinant SEQ ID NO: 19 SEQ ID NO: 19 SEQ ID NO: 19 g.III signal sequence Encoded amino acid sequences Mutated vector Mutated vector Parental (wild-type g.III (wild-type g.III vector, (wild gene encoding gene encoding Encoded type g.III L8P S11P product gene) substitution) substitution) WT g.III gene SEQ ID NO: 1 SEQ ID NO: 21 SEQ ID NO: 22 product Recombinant SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1 g.III gene product c-myc tag SEQ ID NO: 7 SEQ ID NO: 7 SEQ ID NO: 7 WT g.III SEQ ID NO: 18 SEQ ID NO: 18 SEQ ID NO: 18 signal peptide Recombinant SEQ ID NO: 20 SEQ ID NO: 20 SEQ ID NO: 20 g.III signal peptide

TABLE-US-00002 TABLE 2 Mutated, wild- Mutated, wild- Parental, type g.III gene type g.III gene wild type encoding L8P encoding S11P Phage titer g.III gene substitution substitution (pfu/well) (OD.sub.560) (OD.sub.560) (OD.sub.560) 0 0.1364 0.1057 0.137 7.80E+06 0.1473 0.126 0.1434 1.60E+07 0.1449 0.1356 0.1357 3.10E+07 0.1451 0.1381 0.1423 6.30E+07 0.1663 0.1422 0.159 1.30E+08 0.1505 0.1488 0.1711 2.50E+08 0.142 0.1629 0.1887 5.00E+08 0.1777 0.1949 0.2283 1.00E+09 0.1753 0.2347 0.2805 2.00E+09 0.2047 0.4117 0.4519 4.00E+09 0.3157 0.8489 0.9137

[0029] Table 2 provides OD.sub.560 values from a titer-dependent ELISA as generally described above using a PMP/AMP substrate and demonstrates that when using a type 33 M13 phage vector containing mutations that encode L8P or S11P substitutions in the mature, wild type g.III gene product, increased expression of a c-myc protein fused to the P.III surface protein encoded by the recombinant g.III gene is obtained.

EXAMPLE 2

Display of Test Peptide-P.III Fusions on Phage M13 Surface (with Combined 8P+11P Mutations in WT g.III Gene)

[0030] Further modified type-33 bacteriophage M13 vectors comprising mutations which encode both the L8P and S11P substitutions in the mature wild-type g.III gene product (SEQ ID NO:2) are constructed. An exemplary nucleic acid sequence encoding said gene product is given by SEQ ID NO:4. In the same vectors, test peptide-encoding nucleic acid sequences (as given by SEQ ID NOs: 10, 12 and 14, below) are separately cloned in-frame, via linker peptide-encoding sequences, to a c-myc tag-encoding sequence (SEQ ID NO:8) which, in turn, is cloned in frame with and upstream of the recombinant g.III gene sequence (SEQ ID NO:3) in the vector. The same test peptide-encoding nucleic acid sequences are also each separately cloned (in the same manner and format as described above) into the parental type-33 bacteriophage M13 vector which does not comprise either the L8P- or S11P-encoding nucleic acid mutations in the wild type g.III gene (SEQ ID NO:5 provides the nucleic acid sequence of the parental wild type g.III gene without the the L8P- or S11P-encoding mutations).

[0031] Table 3 provides the nucleic acid sequences of the mutated and parental wild-type g.III gene, the recombinant g.III gene, the c-myc tag encoding sequence, the linker peptide encoding sequences and the test peptide-encoding sequences of exemplary vector clones prepared.

[0032] The resulting vectors were used to display the test peptideP.III fusion proteins on the surface of M13 bacteriophage particles harvested from XL-1 blue bacterial cells. The test peptides employed bind with a human IL-6 target protein as further described below. Table 4 provides the corresponding amino acid sequences of the components of the fusion protein products displayed on the harvested M13 phage particles.

TABLE-US-00003 TABLE3 VectorClone NucleicAcid Component 18-24.sup.a 18-22.sup.a 18-4.sup.a Mutatedwildtype (SEQIDNO:4) (SEQIDNO:4' (SEQIDNO:4) g.IIIgene Parentalwildtype (SEQIDNO:5) (SEQIDNO:5) (SEQIDNO:5) g.IIIgene Recombinantg.III (SEQIDNO:3) (SEQIDNO:3) (SEQIDNO:3) gene c-mycencoding (SEQIDNO:8) (SEQIDNO:8) (SEQIDNO:8) sequence Peptidelinker (SEQIDNO:16) (SEQIDNO:16) (SEQIDNO:16) encodingsequence Testpeptide- cgcacctatscaaagaatttg attagcctgtgcgatcagccg ccgccgctgtgcagctggcc encodingsequence gccgctatgtggcggatgaa tatgtgaaaagcctgaacctg ggcgtatcagaaatttggcgg acctattgcgeggcgctg ccgctgtgcccgctggcg cccgctgtgcaccetgggc (SEQIDNO:10) (SEQIDNO:12) (SEQIDNO:14) .sup.aVector clones are prepared with either the mutated, wild type g.III gene containing mutations encoding the L8P and S11P substitutions (i.e. SEQ ID NO: 4), or the parental wild type g.III gene (i.e., SEQ ID NO: 5).

TABLE-US-00004 TABLE4 DisplayedFusionProtein AminoAcid Component 18-24.sup.a 18-22.sup.a 18-4.sup.a Mature,mutatedwild (SEQIDNO:2) (SEQIDNO:2) (SEQIDNO:2) typeg.IIIgene product Mature,parentalwild (SEQIDNO:1) (SEQIDNO:1) (SEQIDNO:1) typeg.IIIgene product Mature,recombinant (SEQIDNO:1) (SEQIDNO:1) (SEQIDNO:1) g.IIIgeneproduct c-mycdetectiontag (SEQIDNO:7) (SEQIDNO:7) (SEQIDNO:7) protein Peptidelinker (SEQIDNO:33) (SEQIDNO:33) (SEQIDNO:33) sequence Testpeptide RTFCKEFGRYVAD ISLCDQPYVKSLNL PPLCSWPAYQKFG sequence ETYCAAL PLCPLA GPLCTLG (SEQIDNO:9) (SEQIDNO:11) (SEQIDNO:13) .sup.aDisplayed fusion proteins contain either the mutated wild type g.III gene product containing the L8P and S11P substitutions (i.e., SEQ ID NO: 2), or the parental wild type g.III gene product.
Harvested phage displaying the test peptideP.III fusion proteins are amplified and titer determination performed as generally described above. Test peptide display levels are then determined by a phage-titer dependent ELISA essentially as described below:

[0033] ELISA plates (Greiner-bio-one, Cat. number: 650061) are coated with 50 l/well of NeutrAvidin (Thermo Scientific, Cat. number: 31050) at 2 g/ml in PBS and allowed to stand overnight at 4 C. Excess sites are blocked by adding 100 l/well of Casein (Thermo Scientific, Cat number: 37528) for one hour at room temperature. 50 l/well of biotinylated human IL6 (R&D Systems, Cat. number 206-IL-010/CF) in PBS is then added to each well and the plates are incubated at room temperature for 30 minutes while rocking. 50 l/well of phage at different titers are then added and the phage diluted in a final concentration of 1% BSA in PBS. Plates are then incubated for 60 minutes at room temperature while rocking. 50 l/well of anti-M13-HRP (G.E., Cat number: 27-9421-01), diluted 1:5,000 in 0.1% tween in PBS is then added followed by incubation of the plates for 60 minutes at room temperature. 50 of Ultra tetramethylbenzidine substrate (Ultra TMB substrate, Thermo Scientific, Cat number: 34029) is then added and the OD at 650 nm is determined.

[0034] Table 5 below provides OD.sub.650 values obtained over a range of phage titers for phage clones 18-24, 18-22 and 18-4, each prepared separately with the type-33 bacteriophage M13 vector encoding the L8P- and S11P-encoding mutations in the wild type g.III gene and the parental type-33 bacteriophage M13 vector (without the L8P- and S11P-encoding mutations in the wild type g.III gene.)

TABLE-US-00005 TABLE 5 Phage Phage Clone 18-24 Phage Clone 18-22 Phage Clone 18-4 titer Parental.sup.a Mutated.sup.b Parental.sup.a Mutated.sup.b Parental.sup.a Mutated.sup.b (pfu/well) (OD.sub.650) (OD.sub.650) (OD.sub.650) (OD.sub.650) (OD.sub.650) (OD.sub.650) 2.3E+08 0.0735 0.3414 0.0536 0.3284 0.0879 0.4286 6.9E+08 0.0426 0.8103 0.0416 0.7749 0.1359 0.988 2.1E+09 0.049 1.4618 0.0535 1.4022 0.3378 1.5715 6.2E+09 0.0521 1.9132 0.0605 1.8236 0.8358 1.918 1.9E+10 0.0675 2.0298 0.1132 1.967 1.4314 2.0064 5.6E+10 0.1189 2.0099 0.3253 1.9578 1.756 1.8853 1.7E+11 0.2946 1.7346 0.7754 1.7187 1.8199 1.5817 5.0E+11 0.6837 1.5017 1.4298 1.4979 1.6572 1.2222 .sup.aPhage clones prepared with type 33 bacteriophage M13 vector containing wild type g.III gene (does not encode L8P and S11P substitutions). .sup.bPhage clones prepared with type 33 bacteriophage M13 vector containing mutated, wild type g.III gene encoding L8P and S11P substitutions.

[0035] The OD.sub.650 values in Table 5 demonstrate that when using a type 33 bacteriophage M13 vector containing mutations that encode L8P and S11P substitutions in the mature, wild type g.III gene product, increased expression of each test peptide fused (via a peptide linker) to the P.III surface protein encoded by the recombinant g.III gene is obtained.

EXAMPLE 3

Display of Fab-P.III Fusions on Phage M13 Surface

[0036] Nucleic acid sequences encoding Fab heavy chain (HC) and light chain (LC) sequences as given by SEQ ID NO:25 and SEQ ID NO:26, respectively, are cloned into a type-33 bacteriophage M13 vector comprising mutations encoding the L8P and S11P substitutions in the mature wild type g.III gene product. The Fab HC-encoding nucleic acid sequence (SEQ 1D NO:27), using a PhoA1 signal peptide-encoding sequence (SEQ ID NO:30), is cloned in-frame and upstream, via a spacer-encoding sequence, to an HA-tag encoding sequence (SEQ ID NO:32) and a c-myc tag encoding sequence (SEQ ID NO:8) which, in turn, are cloned in frame and upstream of the recombinant g.III gene sequence (SEQ ID NO:3) in the vector. The Fab LC-encoding nucleic acid sequence (SEQ ID NO:28) is separately cloned into the vector using a pelB signal peptide encoding sequence (SEQ ID NO:19). Transcription of both the HC and LC Fab-encoding components is under control of a lacZ promoter.

[0037] Double stranded vectors comprising the Fab HC and LC sequences as described above are prepared and used to transfect E.Coli XL1-Blue cells essentially as described previously. The LC sequence (SEQ ID NO:26) is secreted into the bacterial periplasmic space where it forms the Fab dimer with the HC sequence (SEQ ID NO:25) fused via the HA-tag (SEQ ID NO:31) and c-myc tag (SEQ ID NO:7) to the recombinant P.III protein.

[0038] Harvested phage displaying the FabP.III fusion proteins are amplified and titer determination performed as generally described above. Fab display levels are then determined by a phage-titer dependent ELISA using biotinylated TNF as the target ligand as generally described in Nakayama et al., Improving the Copy Number of Antibody Fragment Expressed on the Major Coat Protein of Bacteriophage M13, Immunotechnology, Vol. 12 (1996): 197-207.

[0039] Table 6, below, provides OD.sub.650 values obtained over a range of phage titers for the phage clone displaying the Fab constructs, prepared separately with the type-33 bacteriophage M13 vector encoding the L8P- and S11P-encoding mutations in the wild type g.III gene and the parental type-33 bacteriophage M13 vector (without the L8P- and S11P-encoding mutations in the wild type g.III gene.)

TABLE-US-00006 TABLE 6 Phage Titer Parental.sup.a Phage Titer Mutated.sup.b (pfu/well) (OD.sub.650) (pfu/well) (OD.sub.650) 0 0.0699 0 0.0548 1.69E+06 0.0791 1.69E+05 0.0846 5.08E+06 0.0857 5.08E+05 0.1067 1.52E+07 0.0913 1.52E+06 0.1791 4.57E+07 0.1092 4.57E+06 0.3578 1.37E+08 0.1712 1.37E+07 0.7649 4.12E+08 0.3803 4.12E+07 1.4751 1.23E+09 0.8396 1.23E+08 1.9562 3.70E+09 1.6574 3.70E+08 7.2079 1.11E+10 2.2358 1.11E+09 7.207 3.33E+10 2.4446 3.33E+09 2.0309 1.00E+11 2.5538 1.00E+10 1.3518 .sup.aPhage clones prepared with type 33 bacteriophage M13 vector containing wild type g.III gene (does not encode L8P and S11P substitutions). .sup.bPhage clones prepared with type 33 bacteriophage M13 vector containing mutated, wild type g.III gene encoding L8P and S11P substitutions.

[0040] The OD.sub.650 values in Table 6 demonstrate that when using a type 33 bacteriophage M13 vector containing mutations that encode L8P and S11P substitutions in the mature, wild type g.III gene product, increased expression of a Fab fused (via a peptide spacer) to the P.III surface protein encoded by the recombinant g.III gene is obtained.

TABLE-US-00007 SEQUENCELISTING SEQIDNO:1(maturephageM13surfaceproteinP.III,encodedbyrecombinantand WTg.IIIgene(withoutsignalpeptide)) AETVESCLAKSHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCY GTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPP GTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQY TPVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGG GSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADE NALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDG DNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATF MYVFSTFANILRNKES SEQIDNO:2(mature,mutatedphageM13surfaceproteinP.III(L8P+ S11Pamino acidsubstitutions)encodedbymutatedwild-typeg.III(withoutsignalpeptide)) AETVESCPAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYG TWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPG TEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYT PVSSKAMYDAYWNGKFRDCAFHSGFNEDLYVCEYQGQSSDLPQPPVNAGGGSGGG SGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADEN ALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGD NSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFM YVFSTFANILRNKES SEQIDNO:3(nucleotidesequenceofrecombinantg.IIIgene(withoutsignalpeptide- encodingsequence)) gccgagacagtggagagagcctggccaagttcgcacaccgagaacagatcaccaatgttggaaggatgataagaccctggaccg ctatgccaattacgaaggttgcttatggaacgcaaccggtgtggttgtgtgcacaggcgatgagacccaatgctatggcacctgggtg ccgatcggtctggcaattccggagaacgaaggcggaggtagcgaaggaggtggaagtgaaggcggaggatcggaagggggtgg cacaaagccaccagaatatggagacaccccgattccagatttacacctacattaatccgctggatggtacataccaccaggcaccgaa cagaatccggcaaacccgaacccgagcctggaagaaagccaaccgctgaacacatttatgttccaaaacaaccgttttcgtaaccgtc aaggagccctgaccgtatacaccggtacagtgacccagggtacagatccggtgaagacctactatcaatatacaccggttagcagca aggcaatgtacgatgcatattggaatggcaagtttcgtgattgtgcatttcatagcggtttcaacgaagacctgtttgtgtgcgaatacca gggtcagagcagcgatttaccgcagccaccggttaacgcaggtggtggaagcggagggggaagtggcggtgggtcagaaggcg gaggatcggaaggaggtgggagtgaaggagggggaagcgaaggagggggatcaggaggtggtagcggaagtatgcatacttcata ctacgagaagatggccaatgcaaacaaaggcgcaatgacagagaacgcagacgagaatgcactgcaaagtgatgcaaagggtaa gctggacagcgttgcaaccgactatggagcagcaattgacggctttatcggagatgtcagcggtctggcgaacggcaacggagcaa caggcgacttcgcaggtagcaacagccagatggcacaggttgatagatggcgacaacagtccgctgatataacaactttcgccagtac ctgccgagtctgccacaaagcgtcgagtgccgtccgtttgttttcggtgcaggcaagccgtacgagttcagcatcgactgcgataagat taatctttttcgcggagttttcgcattcctgctgtacgtggcaacgttcatgtacgttttcagcaccttcgccaatatcttacgcaacaaaga aagc SEQIDNO:4(nucleotidesequenceofmutated,wild-typeg.IIIgene(encodingL8P+ S11Paminoacidsubstitution)(withoutsignalpeptide-encodingsequence)) gccataaactgttgaaagttgtccatgcaaaaccccatacagaaaattcatttactaacattctggaaagacgacaaaactttagatcgttac gctaactatgagggctgtctgtggaatgctacaggcgttgtagtttgtactggtgacgaaactcagtgttacggtacatgggttcctattg ggcttgctatccctgaaaatgagggtggtggctctgagggtggcggttctgagggtggcggttctgagggtggcggtactaaacctcc tgagtacggtgatacacctattccgggctatacttatatcaaccctacatacggcacttatccgcctggtactgagcaaaaccccgctaa tcctaatccttctcttgaggagtctcagcctcttaatactttcatgtttcagaataataggttccgaaataggcagggggcattaactgtttat acgggcactgttactcaaggcactgaccccgttaaaacttattaccagtacactcctgtatcatcaaaagccatgtatgacgcttactgga acggtaaattcagagactgcgctttccattaggctttaatgaggatttatttgtttgtgaatatcaaggccaatcgtctgacctgcctcaac ctcctgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggctctgagggtggcggttctgagggtgg cggactgaggatagatcgatttccggtggtggctctggttccggtgattttgattatgaaaagatggcaaacgctaataagatgggctatg accgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgctactgattacggtgagctatcgatgg tttcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacattaatgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgtcgcccttttgtattggcgaggtaaacc atatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcgtttcttttatatgttgccacctttatgtatgtattttctacgttt gctaacatactgcgtaataaggagtct SEQIDNO:5(nucleotidesequenceofwild-typeg.IIIgene(withoutsignalpeptide- encodingsequence)) gccgaaactgttgaaagttgatagcaaaatcccatacagaaaattcattactattgtctggaaagacgacaaaactttagatcgttacg ctaactatgagggctgtctgtggaatgctacaggcgttgtagtttgtactggtgacgaaactcagtattttggtacatgggttcctattgg gcttgctatccctgaaaatgagggtggtggactgagggtggcggttctgagggtggcggttagagggtggcggtactaaacctcct gagtacggtgatacacctattccgggctatacttatatcaaccactcgacggcatttatccgcctggtactgagcaaaaccccgctaat cctaatccttctcttgaatgattctcatcctcttaatactttcatgtttcagaataataggttccgaaataggcagggggcattaactattttata cgggcactgttactcaaggcactgaccccgttaaaacttattaccagtacactcctgtatcatcaaaagccatgtatgacgcttactggaa cggtaaattcagagactgcgctttccattaggctttaatgaggatttatttgtttgtgaatatcaaggccaatcgtctgacctgcctcaacct cagtcaatgaggcggcggctctggtggtggttaggtggcggctctatagatgtatgtatgctctgagggtggcggttagagggtggc ggctctgagggaggcggaccggtggtggctctggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatga ccgaaaatgccgatgaaangcgctacagtctgacgctaaaggcaaacttgattctgtcgctactgattacggtgagctatcgatggtt tcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacggt gataattcacctttaatgaataatttccgtcaatatttaccaccctccctcaatcggttgaatgtcgcccttttgtctttggcgctggtaaacc atatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcgtttatttatatgttgccacctttatgtatgtattttctacgttt gctaacatactgcgtaataaggagtct SEQIDNO:6(nucleotidesequenceforparentaltype-33plasmidwithrecombinant andwTg.IIIgene(withoutL8PandS11Pencodingmutations)(includingsignal peptide-encodingsequences) aatgctactactattagtagaattgatgccaccattcagctcgcgccccaaatgaaaatatagctaattaggttattgaccatagcgaaa tgtatctaatggtcaaactaaatctactcgttcgckataattgggaatcatttgttatatggaatgaaacttccagacaccgtactttagttgc atatttaaaacatgttgagctacagcattatattcagcaattaagctctaagccatctgcaaaaatgacctcttatcaaaaggagcaattaa aggtactctctaatcctgacctattggagtttgcttccgattctggttcgctttgaagctcgaattaaattgcgatatttgaagtctttcgggc ttcctataatctttttatatgcaatccgctttgatctgactataatagtcagggtaaagacctgatttttatatttatggtcattacgttttctgaa ctgataaagcatttgagggggattcaatgaatatttatgacgattccgcagtattggacgctatccagtctaaacattttactgttaccccct ctggcaaaacttcttttgcaaaagcctctcgctattttggtttttatcgtcgtctggtaaacgagggttatgatagtgttgctcttactatgcct cgtaattccttttgatcgttatgtatctatcattagttgaatgtggtattcctaaatctcaactgatgaatattctacctgtaataatgttgttccgtt agttcgattattattgtagatttttcttcccaacgtcctgactggtataatgagccagttcttaaaatcgcataaggtaattcacaatgattaa agttgaaattaaaccatctcaagcccaatttactactcgttctggtgtttctcgtcagggcaagccttattcactgaatgagcagctttgtta cgttgatttgggtaatgaatatccggttcttgtcaagattactcttgatgaaggtcagccagcctatgcgcctggtagtacaccgttcatct gtcctcatcaaagttggtcagttcggttcccttatgattgaccgtctgcgcctcgttccggctaagtaacatggagcaggtcgcggatttc gacacaatttatcagatcgatgatacaaataccgttgtactttattttcgcatcttggtataatcatagggatgtcaaagatgagtgttttagtgt attatttgcctctttcgttttaggttggtgccttcgtagtggcattacgtattttacccgtttaatggaaacttcctcatgaaaaagtattagtc ctcaaagcctctgtagccgttgctaccctcgttccgatgctgtctttcgctgctgagggtgacgatcccgcaaaagcggcctttaactcc ctgcaagcctcagcgaccgaatatatcggttatgcgtgqtcgatggttgttgtcattgtcggcgcaactatcggtatcaagctattttaag aaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttaggagccttttttttggapttttcattgtgaaaaaattatt attcgcaattcattagttgttcctttctattctcactccgccgaaactgttgaaagttgtttagcaaaatcccatacagaaaattcatttactaa cgtctggaaagacgacaaaactttagatcgttacgctaactatgagggctgtagtggaatgctacaggcgttgtagtttgtactggtga cgaaactcagtgttacggtacatgggttcctaagggcttgctatccctgaaaatgagggtggtggctctgagggtggcggttctgagg gtggcggttctgagggtggcggtactaaacctcctgagtacggtgatacacctattccgggctatacttatatcaaccctctcgacggca cttatccgcctatgtactgagcaaaaccccactaatcctaatccttacttgaggagtctcagcctataatactacatgatcagaataata ggttccgaaataggcagggggcattaactgtttatacgggcactgttactcaaggcactgaccccgttaaaacttattaccagtacactc ctgtatcatcaaaagccatgtatgacgcttactggaacggtaaattcagagactgcgctttccattctggattaatgaggatttatagtag tgaatatcaaggccaatcgtctatacctgcctcaacctcctgtcaatgctatgcatgcggctaggtggtggttctggtggcggctctgagg gtagtagctctgagggtggcggttctgagggtggcggctagagggaggcggttccggtggtggctctggttccggtgatatgattat gaaaagatggcaaacgctaataagggggctatgaccgaaaatgccgatgaaaacgcgctacagtagacgctaaaggcaaacttga ttctgtcgctactgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgct ggctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcg gttgaatgtcgcccttttgtattggcgaggtaaaccatatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcattt cttttatatgttgccacattatgtatgtattttctacgtttgctaacatactgcgtaataaggagtcttaatcatgccagttatttgggtattccg ttattattgcgtttcctcggtaccactggtaactagttcggctatctgcttacttacttaaaaagggcttcggtaagatagctattgctatttc attgtttcttgctatattattgggcttaactcaattcttgtatggttatctctctatatattagcgctcaattaccctagactttgttcagggtgttc agttaattctcccgtctaatgcgcttccctgtttttatgttattctctctgtaaaggctgctattttcatttttgacgttaaacaaaaaatcgtttctt atttggattgggataaataatatggctgthattagtaactggcaaattaggctctagaaagacgctcgttagcgaggtaagattcaggat aaaattgtagctgggtgcaaaatagcaactaatcttgatttaaggcttcaaaacctcccgcaagtcgggaggttcgctaaaacgcctcg cgttcttagaataccggataagccttctatatctgatttgcttgctattgggcgcggtaatgattcctacgatgaaaataaaaacggcttgct tatttacgatgagtgcggtacttggtttaatacccatttatgataatgataaggaaagacagccgattattatattggtttctacatgctcgtaa attaggatgggatattatttttcttgttcaggacttatctattgttgataaacaggcgcgttagcattagctgaacatgttgtttattgtcgtcgt aggacagaattactttaccttagtcggtactttatattctcttattactggctcgaaaatgcctagcctaaattacatgttggcgttgttaaat atggcgattacaattaagccctactatttgagcgttggctttatactatgtaagaatttgtataacgcatatatatactaaacagatctttttctag taattatgattccggtgtttattcttatttaacgccttatttatcacacggtcggtatttcaaaccattaaatttaggtcagaagatgaagcttac taaaatatatttgaaaaagattcacgcgttctttgtcttgcgattggatttgcatcagcatttacctttagttatataacccaacctaagccgg aggttaaaaaggtagtactcagacctatgattttgataaattcactattgactatctcagcgtcttaatctaagctatcgctatgttttcaag gattctaagggaaaattaattaatagcgacgatttacagaagcaaggttattcactcacatatattgatttatatactgtttccattaaaaaag gtaattcaaatgaaattgttaaatgtaattaattttgttttcttgatgtttgtttcatcatatcttttgacaggtaattgaaatataataattcgcct ctgcgcgattttgtaacttgg-tattcaaagcaatcaggcgaatccgttattgtttctcccgatgtaaaaggtactgttactgtatattcatag acgttaaacctgaaaatctacgcaatttattatttagttttacgtgcaaatgattttgatatagtaggactaaccatccattattcagaagt ataatccaaacaatcaggattatattgatgaattgccatcatctgataatcaggaatatgatgataattccatctccttaggtggtttattgtt ccgcaaaatgataatgttactcaaacttttaaaattaataacgttcgggcaaaggatttaatacgagttgtcgaattgtttgtaaagtctaata cactaaatcacaaatatattatctattgacggctctaatctattagttgttagtgacctaaagatattttagataaccttcctcaattcctttca actgttgatttgccaactgaccagatattgattatagatgtttgatatttgaatgttcagcaaggtgatgctttagatttttcatttgagctgatctc tcagcgtggcactgttgcaggcggtgttaatactgaccgcctcacctctgttttatcttctgctggtggttcgttcggtatttttaatggcgat gattagggctatcagttcgcgcattaaagactaatagccattcaaaaataagtctgtgccacgtattcttacgattcaggtcagaagggt tctatctctgttggccagaatgtcccttttattactggtcgtgtgactggtgaatctgccaatgtaaataatccatttcagacgattgagcgtc aaaatgtaggtatttccatgagcgtttttcctgttgcaatggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagtt cttctactcaggcaagtgatgttattactaatcaaaataagtattgctacaacggttaatttgcgtgatggacagactcttttactcgattgatcc tcactgattataaaaacacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctgtttagctcccgctctgattct aacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcagcgggtgtggtgg ttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctacttcccttcctttctcgccacgttcgccggatt ccccgtcaagctctaaatcgggggctccattagggttccgatttagtacatacggcacctcgaccccaaaaaacttgatttgggtgatg gttcacgtagtgggccatcgccctgatagacggtattcgccctagacgttggagtccacgactttaatagtggactcttgaccaaactg gaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggaaccaccatcacacaggattttcgcctgct ggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcgctggtga aaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttt cccgactatgaaagcgggcagtgagcgcaacgcaattaatgtgagttagacactcattaggcaccccaggcttgacactttatgcttcc ggctcgtataatgtgtggaattgtgagcggataacaatttcacacgccaaggagacagtcataatgaaatacctattgcctacggcagc cgctggattgttattactcgctgcccaaccagccatggccggcggaggataggcgagcaaaagctcattagtgaagaggatatacc gagacagtatgaatagctgcctatgccaagtcatcacaccgagaacagcttcaccaatgtttggaaggatgataagaccctggaccgcta tgccaattacgaaggttgcttatggaacgcaaccggtgtggttgtgtgcacaggcgatgagacccaatgctatggcacctgggtgccg atcggtctggcaattccggagaacgaaggcggaggtagcgaaggaggtggaagtgaaggcggaggatcagaagggggtggcac aaagccaccagaatatggagacaccccgattccaggttacacctacattaatccgctggatggtacataccctccaggcaccgaaca gaatccggcaaacccaaacccgagcctggaagaaagccaaccgctgaacacatttatgttccaaaacaaccgttacgtaaccgtcaa ggagccctataccgtatacaccggtacagtgacccagggtacagatccggtgaagacctactatcaatatacaccggttaatcagcaag gcaatgtacgatgcatattggaatggcaagtttcgtgattgtgcatttcatagcggtttcaacgaagacctgtttgtgtgcgaataccagg gtcagagcagcgatttaccgcagccaccggttaacgcaggtagtagaagcggagggggaagtggcggtgggtcagaaggcgga ggatcggaaggaggtgggagtgaaggagggggaagcgaaggagggggatcaggaggtggtagcggaagtggcgacttcgact acgagaagatggccaatgcaaacaaaggcgcaatgacagagaacgcagacgagaatgcactgcaaagtgatgcaaagggtaagc tggacagcgttgcaaccgactatggagcagcaattgacggctttatcggagatgtcagcggtctggcgaacggcaacggagcaaca ggcgacttcgcaggtagcaacagccagatggcacaggttggagatggcgacaacagtccgctgatgaacaactttcgccagtacct gccgagtctgccacaaagcgtcgagtgccgtccgtttgttttcggtgcaggcaagccgtacgagttcagcatcgactgcgataagatta atattttcatcggagttttcgcattcctgctgtacgtggcaacgttcatgtacgattcagcaccttcgccaatatcttacgcaacaaagaaa gctaagcaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgattgcctggtttcc ggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgc acggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgtt actcgctcacatttaatgttgatgaaagaggctacaggaaggccagacgcgaattatattgatggcgttcctattggttaaaaaatgag ctgatttaacaaaaatttaatgcgaattttaacaaaatattaacgtttacaatttaaatatttgcttatacaatcttcctgtttttggggcttactg attatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgattacttgtttgaccagactctcaggcaatgacctg atagcctttgtagatctctcaaaaatagctaccctctccggcattaatttatcagctagaacggttgaatatcatattgatggtgatagactg tctccggcattctcacccttttgaatctttacctacacattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatcatgcgt tgaaataaaggatctcccgcaaaagtattacagggtcataatgtttttggtacaaccgatttagctttatgctagaggctttattgataatt ttgctaattattgccttgcagtatgatttattggacgtt SEQIDNO:7(c-mycdetectiontagaminoacidsequence EQKLISEEDL SEQIDNO:8(nucleotidesequenceencodingc-mycdetectiontag) gagcaaaagctcattagtgaagaggatctt SEQIDNO:9(Clone18-24IL-6bindingtestpeptidesequence RTFCKEFGRYVADETYCAAL SEQIDNO:10(nucleotidesequenceencodingClone18-24IL6bindingtestpeptide) aggactttttgtaaggagtttgggcggtatgttgcggatgagacgtattgtgctgcgctt SEQIDNO:11(Clone18-22IL-6bindingtestpeptidesequence) ISLCDQPYVKSLNLPLCPLA SEQIDNO:12(nucleotidesequenceencodingClone18-22IL-6bindingtestpeptide) atttattgtgtgatcagccgtatgttaagagtcttaatcttccgttgtgtccgcttgct SEQIDNO:13(Clone18-4iL-6bindingtestpeptidesequence) PPLCSWPAYQKFGGPLCTLG SEQIDNO:14(nucleotidesequenceencodingClone18-4IL-6bindingtestpeptide) cctccgctgtgttcaggcctgcaatcagaagtttggtggtccgctgtgtacgcttggt SEQIDNO:15(nucleotidesequencefortype-33plasmidwithrecombinantg.IIIand mutatedWTg.IIIgene(withL8PandS11Pencodingmutations)(includingsignal peptide-encodingsequences) aatgctactactattagtagaattgatgccaccttttcagctcgcgccccaaatgaaaatatagctaaacaggttattgaccatttgcgaaa tgtatctaatggtcaaactaaatctactcgttcgcagaattgggaatcaactgttatatggaatgaaacttccagacaccgtactttagttgc atatttaaaacatgttgagctacagcattatattcagcaattaagttttaagccatctgcaaaaatgacctatatcaaaaggagcaattaa atgtactctctaatcctgacctgttggagtttgatccatgtaggttcatcatgaagctcgaattaaaacgcgatatttgaagtctttcatggc ttcctcttaatattttgatgcaatccgattgcactgactataatagtcagggtaaagacctgatttttgatttatggtcattctcgttttctgaa ctgtttaaagttatttgagggggattcaatgaatatttatgacgattttcaagtattggattgtttatccagtctaaacattttactgttaccccct aggcaaaacttcattgcaaaagcctacgctattaggtattatcgtcgtaggtaaacgagggttatatatagtgagctcttactatgcct cgtaattccattggcgttatgtatctgcttttagttgaatgtggtattcctaattttttcaactgatgaatctttctacctgtatttaatgttgttccgtt agttcgttttattaacgtagatttttcttcccaacgtcctgactggtataatgagccagttcttaaaatcgcataaggtaattcacaatgattaa agttgaaattaaaccatctcaagcccaatttactactcgactggtgtttctcgtcagggcaagccttattcactgaatgagcagctttgtta cgttgatttgggtaatgaatatccggttcttgtcaagattactcttgatgaaggtcagccagcctatgcgcctggtctgtacaccgttcatct gtcctctttcaaaatttggtcagttcggttccatatgattgaccgtctgcgcctcgttccatgctaagtaacatatgatcaggtcgcggatttc gacacaatttatcaggcgatgatacaaatctccgttgtactttgtttcgcgcttggtataatcgagggggtcaaagatgagtgttttagtgt attcttttgcctcatcgttttaggttggtgccttcgtagtggcattacgtattttacccgtttaatggaaacttcctcatgaaaaagtctttagtc ctcaaagcactgtagccgttgctaccacgttccgatgctgtctttcatctgagagggtgacatatcccgcaaaagcgatcctttaactcc ctgcaagcctcagcgaccgaatatatcggttatgcgtgggcgatagttgttgtcattgtcggcgcaactatcggtatcaagctgataag aaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagccttttttttggagattttcaacgtgaaaaaattatt attcgcaattcctttagttgttcattctattacactccgccgaaactgttgaaagttgtccggcaaaaccccatacagaaaattcatttact aacgtctggaaagacgacaaaactttagatcgttacgctaactatgagggctgtctgtggaatgctacaggcgagtagatgtactggt gacgaaactcagtgttacggtacatgggttcctattgggcttgctatccctgaaaatgagggtagtggctagagggtggcggttctga gggtggcggttctgagggtggcggtactaaacctcctgagtacggtgatacacctattccgggctatacttatatcaaccctacgacg gcacttatccgcctggtactgagcaaaaccccgctaatcctaatccactcttgaggagtctcagcctcttaatactttcatgatcagaata ataggttccgaaataggcagggggcattaactgtttatacgggcactgttactcaagatcactgaccccgttaaaacttattaccagtaca ctcctgtatcatcaaaagccatgtatgacgcttactggattggtaaattcagagactgcgattccattctggctttaatgaggatttatttgt ttgtgaatatcaaggccaatcgtctgacctgcctcaacctcctgtcaatgctggcggcggctctggtggtggttctggtagcggctctga gggtggtggctctgagggtggcggttctatagatgtatgcatgctctgagggaggcggttccggtggtggactggttccggtgattttgat tatgaaaagatggcaaacgctaataagggggctatgaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaactt gattctatcgctactgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttg ctggctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatc ggttgaatgtcgcccttttgtctttggcgctggtaaaccatatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcgtt tcttttatatgttgccacctttatgtatgtattttctacgtttgctaacatactgcgtaataaggagtcttaatcatgccagttcttttgggtattcc gttattattgcgtttcctcggtttccttctggtaactttgttcggctatagcttacttttcttaaaaagggatcggtaagatagctattgctattt cattgtttcttgctcttattattgggcttaactcaattcttgtgggttatctctctgatattagcgctcaattaccctctgactttgttcagggtgtt cagttaattctcccgtctaatgcgcttccctgtttttatgttattactagtaaaggagctattttcatttagacgttaaacaaaaaatcgtttc ttatttggattgqtataaataatatggagtttattttgtaactggcaaattaggctctggaaagacgctcgttagcgttggtaagattcaatg ataaaattgtagctgggtgcaaaatagcaactaatcttgatttaaggcttcaaaacctcccgcaagtcgggaggttcgctaaaacgcctc gcattatagaataccggataagccttctatatagatttgcttgctattgggcgcggtaatgattcctacgatgaaaataaaaacggcttg cttgttctcgatgagtgcggtacttggtttaatacccgttcttggaatgataaggaaagacagccgattattgattggtttctacatgctcgt aaattaggatgggatattattUtcttgttcaggacttatctattgttgataaacaggcgcgttctgcattagctgaacatgagtttattgtcgt cgtctgatacagaattactttaccttttgtcggtactttatattctcttattactggctcgaaaatgcctctgcctaaattacatgttggcgttgtt aaatatggcgattctcaattaagccctactgttgagcgttggattatactggtaagaatttgtataacgcatatgatactaaataggatttt ctagtaattatgattccggtgtttattatatttaacaccttatttatcacacggcggtatttcaaaccattaaatttaggtcagaagatgaag cttactaaaatatatttgaaaaagttttcacgcgttctttgtcttgcatattggatttgcatcagcatttacatatagttatataacccaacctaag ccggaggttaaaaaggtagtctctcagacctatgattttgataaattcactattgactcttctcagcgtcttaatctaagctatcgctatgtat caaggattctaagggaaaattaattaatagcgacgatttacagaagcaaggttattcactcacatatattgatttatgtactgtttccattaaa aaaggtaattcaaatgaaattgttaaatgtaattaattttgttttcttgatgtttgtttcatcatcttatttgctcaggtaattgaaatgaataattc gcctagcgcgattagtaacttggtattcaaagcaatcaggcgaatccgttattgtttctcccgatgtaaaaggtactgttactgtatattca tctgacgttaattctgaaaatctacgcaatttctttatttctgattacgtatcaaatatattttgatatatgtaggttctaacccttccattattcaga agtataatccaaacaatcaggattatattgatgaattgccatcatctgataatcaggaatatgatgataattccgctccttaggtggtttatt gttccgcaaaatgataatgttactcaaacttttaaaattaataacgttcgggcaaaggatttaatacgagttgtcgaattgtagtaaagtcta atacttctaaatcacaaatattattatctattgacatgctctaatctattagttgttagtatctcctaaagatattttagataaccttcctcaattcctt tcaactgttgatttgccaactgaccagatattgattgagggtttgatatttgaggttcagcaaggtgatgctttagatttttcatttgctgctgg ctctcagcgtggcactgttgcaggcggtgttaatactgaccgcctcacctctgttttatcttctgctggtggttcgttcggtatttttaatggc gatgttttagggctatcagttcgcgcattaaagactaatagccattcaaaaatattgtagtgccacgtattcttacgctttcaggtcagaag ggttctatctctgttggccagaatgtcccttttattactggtcgtgtgactggtgaatctgccaatgtaaataatccatttcagacgattgagc gtcaaaatgtagattatttccatgagcgtattcctgttgcaatggctggcggtaatattgttctatgatattaccagcaaggccgatagtttga gttcttctactcaggcaagtgatgttattactaatcaaagaagtattgctacattggttaatttgcgtgatggacagactcttttactcggtg gcctcactgattataaaaacacttacaggattaggcgtaccgttcagtctaaaatccctttaatcggcctcagtttagctcccactag attctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcatgcatggtgtat gtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccactcctttcgctacttccatccatctcgccacgttcgccg gctttccccgtcaagctctaaatcgggggaccattagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttggg tgatggttcacgtagtgqtccatcgccctgatagacggtttttcgccctttgacgttgatagtccacgttctttaatagtggactcttgttcca aactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggaaccaccatcacacaggattttcg cdactggggcaaaccagcatggaccgcttgaacaactctctcagggccaggcggtgaagggcaatcagctattgcccgtctcgct ggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctaccccgcgcgttggccgattcattaatgcagctggcacgac aggtttcccgactggaaagcgggcagtgagcgcattgcaattaatgtgagttagctcactcattaggcaccccaggcttgacactttat gatccggctcgtataatgtgtggaattgtgagcggataacaatttcacacgccaaggagacattcataatgaaatacctattgcctacg gcagccgaggattgttattactcgagcccaaccagccatggccggcggaggatctggcgagcaaaagctcattagtgaagaggat cttgccgagacagtggagagctgcctggccaagtcgcacaccgagaacagcttcaccaatgtttggaaggatgataagaccagga ccgctatatccaattacgaagatttgatatggaacatcaaccatgattgatttgtgtgcacaggcgatgagacccaatgctatggcacctgat gtgccgatcggtctggcaattccggagaacgaaggcggaggtagcgaaggaggtggaagtgaaggcggaggatcggaaggggg tggcacaaagccaccagaatatggagacaccccgattccaggRacacctacattaatccgaggatggtacataccctccaggcacc gaacagaatccggcaaacccgaacccgagcctggaagaaagccaaccgctgaacacatttatgttccaaaacaaccgttttcgtaac cgtcaaggagccctgaccgtatacaccggtacagtgacccagggtacagatccggtgaagacctactatcaatatacaccggttagc agcaaggcaatgtacgatgcatattggaatggcaagtttcgtgattgtgcatttcatagcggtttcaacgaagacctgtttgtgtgcgaat accagggtcagagcagcgatttaccgcagccaccggttaacgcaggtggtggaagcggagggggaagtggcggtgggtcagaag gcggaggatcggaaggaggtgagagtgaaggagggggaagcgaaggagggggatcaggaggtggtagcggaagtggcgactt cgactacgagaagatggccaatgcaaacaaaggcgcaatgacagagaacgcagacgagaatgcactgcaaagtgatgcaaaggg taagctggacagcgttgcaaccgactatggagcagcaattgacggctttatcggagatgtcagcggtctatgcgaacggcaacggag caacaggcgacttcgcaggtagcaacagccagatggcacaggttggagatggcgacaacagtccgctgatgaacaactttcgccag tacctgccgagtctgccacaaagcgtcgagtgccgtccgtttgttttcggtgcaggcaagccgtacgagttcagcatcgactgcgataa gattaatctUttcgcggagttttcgcattcctgctgtacgtggcaacgttcatgtacgttttcagcaccttcgccaatatcttacgcaacaaa gaaagctaagcaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctgg tttccggcaccagaagcggtgccggaaagaggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactatgcag atgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacggg ttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttcctattggttaaaaaat gagctgatttaacaaaaatttaatgcgaattttaacaaaatattaacgtttacaatttaaatatttgatatacaatcttcctgtttttggggctttt ctgattatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgattctcttgtttgctccagactctcaggcaatgac ctgatagcctttgtagatctctcaaaaatagctaccctctccggcattaatttatcagctagaacggttgaatatcatattgatggtgatttga ctgtctccggcattctcacccttttgaatctttacctacacattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatcatg cgttgaaataaaggcttctcccgcaaaagtattacagggtcataatgtttttggtacaaccgatttagctttatgctctgaggctttattgctt aattttgctaattctttgccttgcctgtatgatttattggacgtt SEQIDNO:16(nucleotidesequenceencodingClone18-24,18-22,and18-4peptide linker) ggcggaggataggc SEQIDNO:17(nucleotidesequenceencodingendogenousbacteriophageM13P.III signalpeptide) gtgaaaaaattattattcgcaatcctttagttgttcattcattctcactcc SEQIDNO:18(aminoacidofendogenousbacteriophageM13P.IIIsignalpeptide) VKKLLFAIPLVVIPFYSHS SEQIDNO:19(nucleotidesequenceencodingpelBsignalpeptide) atgaaatacctattgcctacggcagccgctggattgttattactcgctgcccaaccagccatggcc SEQIDNO:20(pelBsignalpeptide) MKYLLPTAAAGLLLLAAQPAMA SEQIDNO:21(aminoacidsequenceofmatureWTg.IIIgeneproductcomprisingL8P substitution)(withoutsignalpeptide)) AETVESCPAKSHTENSFTNNWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYG TWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPG TEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYT PVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGGG SGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADEN ALQSDAKCALDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGD NSPLMNNFRQYLPSLPQSVECRPFVFGAGKRYEFSIDCDKINLFRGVFTAFLLYVATFM YVFSTFANILRNKES SEQIDNO:22(aminoacidsequenceofmatureWTg.IIIgeneproductcomprising S11Psubstitution)(withoutsignalpeptide) AETVESCLAKPHTENSFTNWVKDDKTLDRYANYEGCLWNATGVVVCTGDETQCY GTWVPIGLAIPENEGGGSEGGSEGGGSEGGGTKPPYGDTPIPGYTYINPLDGTYPP GTEWNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQY TPVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGG GSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENAD NALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDG DNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATF MYVFSTFANILRNIKES SEQIDNO:23(nucleotidesequenceencodingmature,WTg.IIIgeneproduct comprisingL8Psubstitution(withoutsignalpeptide) gccgaaactgttgaaagttgtccggcaaaatcccatacagaaaattcatttactaacgtctggaaagacgacaaaactttagatcgttac gctaactatgagggctgtctgtggaatgctacaggcgttgtagtttgtactggtgacgaaactcagtgttacggtacatgggttcctattg ggcttgctatccctgaaaatgagggtggtggactgagggiggcggttcatagggatgcg,gttagagggtggcggtactaaacctcc tgagtacggtgatacacctattccgggctatacttatatcaaccctctcgacggcacttatccgcctggtactgagcaaaaccccgctaa tcctaatccttctcttgaggagtctcagcctcttaatactttcatgtttcagaataataggttccgaaataggcagggggcattaactgtttat acgggcactgttactcaaggcactgaccccgttaaaacttattaccagtacactcctgtatcatcaaaagccatgtatgacgcttactgga acggtaaattcagagactgcgctttccattctggctttaatgaggatttatttgtttgtgaatatcaaggccaatcgtctgacctgcctcaac ctcctgtcaatgaggcggcggctaggtggtggttctggtggcggctctgtagggtgtgtggctctgagggtggcggactgagggtgg cggctctgagggaggcggttccggtggtggctctggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatg accgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgctactgattacggtgctgctatcgatgg tttcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgtcgcccttttgtctttggcgctggtaaacc atatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcgtttcttttatatgttgccacctttatgtatgtattttctacgttt gctaacatactgcgtaataaggagtct SEQIDNO:24(nucleotidesequenceencodingmature,WTg.IIIgeneproduct comprisingS11Psubstitution(withoutsignalpeptide) gccgaaactgtataaagtattttagcaaaaccccatacagaaaattcatttactaacgtaggaaagacgacaaaactttagatcgttacg ctaactatgagggctgtctgtggaatgctacaggcgttgtagtttgtactggtgacgaaactcagtgttacggtacatgggttcctattgg gcttgctatccctgaaaatgagggtggtggctctgagggtggcggttctgagggtggcggttctgagggtggcggtactaaacctcct gagtacggtgatacacctattccgggctatacttatatcaaccctctcgacggcacttatccgcctggtactgagcaaaaccccgctaat cctaatccttctcttgaggagtctcagcctcttaatactttcatgtttcagaataataggttccgaaataggcagggggcattaactgtttata cgggcactgttactcaaggcactgaccccgttaaaacttattaccagtacactcctgtatcatcaaaagccatgtatgacgcttactggaa cggtaaattcagagactgcgattccattctggctttaatgaggatttatttgtttgtgaatatcaaggccaatcgtagacctgcctcaacct cctgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggctctgagggtggcggttctgagggtggc ggctctgagggaggcggttccggtggtggctctggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatga ccgaaaatgccgatgaaaacgcgctacagtagacgctaaaggcaaacttgattctgtcgctactgattacggtgagctatcgatggtt tcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacggt gataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgtcgcccttttgtctttggcgaggtaaacc atatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgtttcttttatatgttgccdcattatgtatgtattactacgat gctaacatactgcgtaataaggagtct SEQIDNO:25(Fab_HCaminoacidsequence) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTADDLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC SEQIDNO:26(Fab_LCaminoacidsequence) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:27(nucleotidesequenceencodingFab_HC) gaggtgcagctggtggagtctgggggaggcttggtacagcctgggaggtccctgagactctcctgtgcagcctctggattcacctttg atgactatgccatgcactgggtccgccaggctccagggaaggggctggagtgggtgtcagctattacaggaatagtggtcacataga ctacgcagactccgtggagggccggttcaccataccagagacaatgccaagaactccctgtatagcaaatgaacagcagagagc cgaggacacggccgtatattactgtgcgaaagtgagctacctgagtactgaaccagcctggactactggggccaaggaaccttggt caccgtctcctcagcctccaccaagggcccatcggtcttccccaggcaccacctccaagagcacctctgggggcacagcggccct gggctgcctsgtcaaggactacaccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccncc cggagtcctacagtartcaggactctactccctcagcagcgtggtgaccgtgccaccagcagcttgggcacccagacctacatctg caacgtgaatcacaagcccagcaacaccaaggIggacaagaaagcagagcccaaatcagc SEQIDNO:28(nucleotidesequenceencodingFab_LC) gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcattc gcaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactagcaatcaggggtcccat ctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcatt gctataaccgtgccccttacacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtatcatcttcccg ccatctgatgagcagagaaatctggaacagcctctgagtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagca ccctgacgctgagcaaagcagactacgagaanacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca aagagcttcattaggggagagtgc SEQIDNO:29(PhoAsignalpeptide) VKQSTIALALLPLLFTPVAKA SEQIDNO:30(nucleotidesequenceencodingPhoAsignalpeptide) gtgaaacaaagcactattgcactggcactcttaccgttactgtttacccctgtcgcaaaagcc SEQIDNO:31(HAtagpeptide) YPYDVPDYAS SEQIDNO:32(nucleotidesequenceencodingHAtagpeptide) tacccgtacgacgttccggattatgccagc SEQIDNO:33(Clone18-24,18-22,and18-4peptidelinkeraminoacidsequence) GGGSG SEQIDNO:34(linkerpeptide) AEAAAKEAAAKEAAAKA SEQIDNO:35(linkerpeptide) AEAAAKEAAAKEAAAKAGGGGS SEQIDNO:36(linkerpeptide) ALAAAKEAAAKEAAAKAGPPGP