Modified mussel proteins, uses thereof and related compounds

Abstract

Disclosed is a mussel adhesive protein including at least one photocaged 3,4-dihydroxyphenylalanine derivative residue including a protecting group on at least one hydroxyl residue of its catechol moiety. The photocaged 3,4-dihydroxyphenylalanine derivative residue replaces a naturally occurring amino acid and the protecting group can be cleaved from the 3,4-dihydroxyphenylalanine derivative residue by irradiation with UV light.

Claims

1. A modified mussel adhesive foot protein-5 (fp-5), comprising a plurality of ortho-nitrobenzyl-3,4-dihydroxyphenylalanine residues, wherein each ortho-nitrobenzyl-3,4-dihydroxyphenylalanine residue comprises an ortho-nitrobenzyl group, wherein each ortho-nitrobenzyl-3,4-dihydroxyphenylalanine residue replaces a tyrosine residue and wherein the ortho-nitrobenzyl group can be cleaved from the ortho-nitrobenzyl-3,4-dihydroxyphenylalanine residue by irradiation with UV light, wherein the modified mussel adhesive fp-5 comprises an amino acid sequence being at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

2. The modified mussel adhesive protein according to claim 1, comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

3. The modified mussel adhesive protein according to claim 2, comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 4 that is fused to the N-terminus of the amino acid sequence defined in claim 2.

4. A nucleic acid encoding for a modified mussel adhesive protein according to claim 3, having a sequence being at least 99% identical to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

5. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 1.

6. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 2.

7. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 3.

8. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 11.

9. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 12.

10. The modified mussel adhesive foot protein according to claim 1, wherein the modified mussel adhesive foot protein has the sequence of SEQ ID NO: 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Aspect of the solution will be explained in more detail in the following making reference to exemplary embodiments and to accompanying Figures.

(2) FIG. 1A shows an SDS-PAGE and Western Blot analysis of MBP-fp-5 variants expressed in B95.ΔA23;

(3) FIG. 1B shows an NBT staining of fp-5 variants expressed in presence of m-ONB-Dopa;

(4) FIG. 1C shows a surface-coating analysis under dry conditions;

(5) FIG. 2 shows a deconvoluted ESI-MS spectrum of MBP-fp-5(5TAG) after incorporation of m-ONB-Dopa;

(6) FIG. 3A shows a MALDI-TOF spectrum of MBP-fp-5(5TAG);

(7) FIG. 3A shows a MALDI-TOF spectrum of MBP-fp-5(10TAG);

(8) FIG. 4A shows F-D curves of fp-5(5TAG) interacting with a mica surface, obtained by an AFM analysis; and

(9) FIG. 4B shows force values of two functionalized tips with fp-5 WT and with fp-5(5TAG), obtained by AFM analysis.

DESCRIPTION OF THE INVENTION

First Exemplary Embodiment

(10) Many generally known protecting groups can be used to produce a protected 3,4-dihydroxyphenylalanine derivative and thus to allow spatiotemporal activation of Dopa's adhesive properties.

(11) An elegant strategy involves engineering the metabolism of bacterial cells in order to produce protected L-Dopa analogues from easily available, cheap precursor molecules. To convert these precursors into amino acids, recombinant strains can be created which express a novel engineered phenylalanine-ammonia lyase (PAL) or tyrosine-ammonia lyase (TAL).

(12) O-pairs, e.g. based on MjTyrRS, are designed for in vivo tRNA aminoacylation with these protected L-Dopa derivatives. Deprotection can be achieved via different ways such as light-exposure or, as shown in the following reaction scheme 1, via acidic hydrolysis, finally leading to an underwater adhesive protein.

(13) ##STR00003##

Second Exemplary Embodiment

(14) ONB-Dopa was used as protected (photocaged) 3,4-dihydroxyphenylalanine derivative residue throughout this example.

(15) To test whether multi-site incorporation of ONB-Dopa (to be more specifically, the ONB group was attached at the meta hydroxyl group of the catechol moiety; m-ONB-Dopa) into proteins naturally displaying high Dopa contents is feasible, a MAP type 5 (fp-5) was chosen as fp-5 is key component of the wet adhesion abilities of mussels. Fp-5 displays the highest Dopa contents of ˜30 mol % which makes it especially attractive for multi-site incorporation of Dopa analogs. For expression tests, a fusion construct was used consisting of an N-terminal maltose binding protein (MBP) sequence with an additional TEV cleavage site and a C-terminal fp-5 sequence from M. galloprovincialis equipped with a His.sub.6 tag.

(16) Tyrosine codons were replaced at five or ten positions with amber codons to allow site-specific incorporation of m-ONB-Dopa by means of a novel ONB-Dopa-specific aaRS (ONB-DopaRS-1, SEQ ID NO: 8). For protein expression, the E. coli BL21(DE3) strain derivative B-95.ΔA23 was chosen, in which RF1 is eliminated. SDS-PAGE and Western blotting indicate the incorporation of m-ONB-Dopa into fp-5(5 amber codons; 5TAG) and fp-5(10 amber codons; 10TAG). The results are shown in FIG. 1A. WT MBP-fp-5 (lane 1), MBP-fp-5(5TAG) (lane 3), and MBP-fp-5(10TAG) (lane 5) were digested with TEV protease and insoluble fractions of fp-5 (lane 2), fp-5(5TAG) (lane 4), and fp-5(10TAG) (lane 6) were analyzed. Depending on the ONB-Dopa content, the expected molecular weight of MBP-fp-5 variants is ˜51-54 kDa and ˜10-12 kDa for fp-5 variants after TEV digest.

(17) The occurrence of multiple bands of purified ONB-Dopa containing fp-5(5TAG) and fp-5(10TAG) variants in SDS PAGE analysis might be caused by partial reduction of the nitro group of ONB to an amine as previously reported..sup.21 Approximately ˜6 mg l.sup.−1 and ˜1 mg l.sup.−1 of purified fp-5(5TAG) or fp-5(10TAG) were obtained in presence of m-ONB-Dopa, respectively, compared to ˜18 mg l.sup.−1 of wild-type (WT) fp-5 (containing 19 Tyr residues).

(18) Production and decaging of fp-5(5TAG) and fp-5(10TAG) variants bearing ONB-Dopa was verified after TEV digest by employing the redox-cycling nitro blue tetrazolium (NBT), which selectively stains Dopa or Dopaquinone containing proteins..sup.22 While pronounced staining occurred in irradiated (+) Fp-5(5TAG) and Fp-5(10TAG) samples, with the latter showing stronger staining, almost no color development was observed without irradiation (−) (FIG. 1B). This indicates successful decaging of ONB-Dopa by UV irradiation.

(19) As a proof-of-principle test for Dopa-mediated adhesion, the surface adhesion ability of fp-5 variants was tested using a direct surface coating assay under dry conditions.sup.11 (FIG. 1C). The upper panel of FIG. 1C shows an image of Coomassie-stained dots. Equal amounts of bovine serum albumin (BSA) and fp-5 variants were spotted at least six times with (+) or without (−) irradiation at 365 nm onto a polystyrene surface. The quantification of dot intensities shown in the lower panel of FIG. 1C indicates elevated adhesive potential after irradiation. The data represent mean±s.d.

(20) The obtained data indicate elevated adhesion on polystyrene surfaces with increasing Dopa content after UV irradiation, demonstrating the adhesive potential of recombinantly produced photocaged mussel proteins. Taken together, these results show that ONB-DopaRS-1 facilitates efficient multi-site incorporation of ONB-Dopa into mussel protein fp-5, thus allowing recombinant production of photocaged MAPs with adhesive potential.

(21) The properties of the produced proteins were further analyzed by mass spectrometry (FIGS. 2 and 3). FIG. 2 shows a deconvoluted ESI-MS spectrum of MBP-fp-5(5TAG) after incorporation of m-ONB-Dopa using ONBYRS-1. The found and expected masses are as follows: MBP-fp-5 (5 ONB-Dopa), observed: 52114.7 Da, expected: 52115.8 Da.

(22) FIG. 3A shows a MALDI-TOF spectrum of MBP-fp-5(5TAG) and FIG. 3B shows a MALDI-TOF spectrum of MBP-fp-5(10TAG) after incorporation of m-ONB-Dopa, TEV digest and irradiation with UV light. The found and expected masses are as follows: fp-5(5 Dopa), observed: 9807.9 Da (M+H.sup.+), expected: 9807.7 Da (M+H.sup.+). fp-5(10 Dopa), observed: 9887.5 Da (M+H.sup.+), expected: 9887.7 Da (M+H.sup.+).

(23) In order to demonstrate the underwater adhesive potential of photocaged MAPs, atomic force microscopy (AFM) based force spectroscopy was employed which has been used to study Dopa-mediated wet adhesion. For this purpose, a bifunctional acetal-polyethylenglycol (PEG)-N-hydroxy-succinimide (NHS) linker molecule.sup.24,25 allowed covalent attachment of MAPs via lysine residues.

(24) Force-distance (F-D) curves of functionalized AFM tips were measured in sodium acetate buffer (10 mM, pH 4.6) on mica surfaces before and after irradiation with UV light (see FIGS. 4A and 4B). FIG. 4A depicts both approach and retraction signals. FIG. 4B shows force values of two tips (a, b) functionalized with different fp-5 variants (namely, fp-5 WT, fp-5(5TAG), and fp-5(10TAG)) before (white bars) and after irradiation (black bars). Data represent mean±s.d. of 100 F-D curves; significance is designated by symbols *p<10.sup.−3, **p<10.sup.−6, ***p<10.sup.−6. While adhesion forces of fp-5 WT did not change significantly through UV irradiation in any measurement, fp-5(5TAG) and fp-5(10TAG) showed a significant increase of the adhesion force (up to 12-fold or up to 6.5-fold, respectively) upon UV light exposure.

(25) To verify that Dopa accounts for the increased adhesion, unmodified and amino-functionalized tips were investigated. Both showed adhesion in the low pN range, in each case unaffected from UV light exposure. The data of fp-5 equipped with five or ten instances of m-ONB-Dopa provide clear evidence for the feasibility of spatiotemporal control of Dopa-mediated adhesion and the high potential of recombinantly produced photocaged MAPs.

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