Title: Self-assembling of Chimeric Mussel-inspired Bio-adhesives originated from Mytilus Californianus and Anabaena flos-aquae : A New Approach to Develop Underwater Adhesion

: Bio-adhesives play a pivotal role in a wide range of medical applications. However, there are some problems about their application in varied pH values and low adhesion force under wet conditions. Here, we report new recombinant fusion protein achieved by mussel foot proteins (Mfps) of Mytilus Californianus and gas vesicle protein A (GvpA) of Anabaena flos-aquae by genetic engineering methods . These chimeric proteins self-assembled into ß-sheet rich fibres because of GvpA amyloid structure. Also, their adhesion forces were significantly increased especially in alkaline environment based on Mfp-3 and Mfp-5. This study illustrates that copolymer of Mfp-5-GvpA:GvpA-Mfp-3 can be used as an underwater sturdy adhesive with tolerance to auto-oxidation, especially at basic conditions.


Introduction:
Naturally, bio-inspired adhesives originated from mussel, barnacles, Notaden frogs and gecko have had extensive interest among scientists due to being water-based adhesive 1-5 . On the one hand, the presence of large quantities of Dihydroxyphenylalanine (DOPA) known to encourage adhesion on wet surfaces 6  On the other hand, amyloids as bio-inspired adhesives can make novel opening for surface/interface functionalization 9 . The versatility of amyloids have attracted the attention of scientists 10 . In a significant number of pathologies, including neurodegenerative diseases and systemic amyloidosis, amyloid fibrils have been identified 11 . A Polypeptide molecules capable of self-assembly into ß-sheet rich linear aggregates are amyloidogenic proteins 12 . A β-hairpin that forms an antiparallel β-sheet is found in amyloid monomers. Amyloids are versatile proteins for interfacial underwater adhesion. For interfacial underwater adhesion, such fibrillar structures have incredible advantages 13 . A study was conducted regarding the presence of amyloid fibers in Gas vesicle protein A (GvpA) of Anabaena flos-aquae. This protein has some advantages such as tolerance to proteolysis, providing the strength of protein structure and amphiphilic properties 14 .
In our study, we have logically planned for a novel chimeric bioadhesive involving musselinspired adhesive and amyloidogenic protein. For having superglue, Mfp-3 and Mfp-5 have been selected with highest Tyrosine (DOPA-based mussel foot protein) among other Mfps and GvpA single protein as amyloidogenic protein has been chosen from Gas Vesicle wall of Anabaena flosaquae. Also, Mfp-5-GvpA and GvpA-Mfp-3 were built. Due to having self-assembly of GvpA as amyloidogenic protein, it is expected that both Mfp-5-CsgA and CsgA-Mfp-3 could self-assemble together into amyloid fibrils with adhesive features by showing DOPA residue on the outside of amyloid scaffold to create copolymers according to previous research work ) Figure 1) 4 . They can also build more strength, resistant to proteolysis and protein flexibility and elasticity, achieved by GvpA in chimeric protein 14 . moreover, we have measured force adhesion between these functional chimeric proteins and mica under wet conditions. The impact of varied pH values on adhesion has been investigated too. We have reached maximum force adhesion at neutral pH and found that copolymers at alkaline pH had the most force adhesion. Nonetheless, other approaches have failed to attain underwater adhesion at neutral pH 15,16 .

Results and discussion:
Expression of recombinant Mfp-5-GvpA and recombinant GvpA-Mfp-3 hybrids: The results illustrated in this research work match the state-of-the-art chimeric proteins. Formerly, Mfp-5-GvpA and GvpA-Mfp-3 cDNAs were specifically optimized for E. coli (Mfp-3 and Mfp-5 genes from Mytilus californianus and GvpA gene from Anabaena flos-aquae), synthesized (by Biomatik Co., Canada) and inserted into a pET-11a expression vector, and then were transformed into E. coli BL21 (DE3). Induction of Mfp-5-GvpA and GvpA-Mfp-3 recombinant hybrids were performed by IPTG 1mM but there are not expressed proteins in SDS-PAGE and Western Blotting, which can be due to toxicity of produced recombinant proteins 17,18 . Hence, E. coli BL21-AI have been used as alternative for resolving this problem 19,20 . After the induction of aforementioned recombinant mussel-inspired amyloid hybrids with L-arabinose 0.2% and IPTG 1mM, cells were  (Table 1) were added. Pellets are resuspended afterwards. A total of 100 mM PMSF and then 10 mg/ml lysozyme (Sigma) were added. The whole suspension was stirred. After that deoxycholic acid were subjoined. The suspension was stored at 37°C while stirring sporadically. When Lysate became viscous, 1 mg/ml DNase was added. Lysates have been incubated at 4°C overnight.

SDS-PAGE and Western Blotting
Initially, samples were mixed with 1x SDS loading buffer (50 mM Tris-Cl (pH 6.8), 100 mM dithiothreitol, 2% (w/v) SDS, 0.1% bromophenol blue, and 10% (v/v) glycerol), and they were then boiled. SDS-PAGE was prepared with 15% separating gel and 5% stacking gel. After running the SDS-PAGE, the gels were stained with Coomassie Brilliant Blue. Having samples run on 15% SDS-polyacrylamide gels, they were transferred onto Nitrocellulose membrane. Chimeric proteins were marked by Monoclonal Anti-polyHistidine-Peroxidase (Sigma, A7058) at a dilution of 1:2000. The expressed proteins were confirmed using H2O2, and diaminobenzidine generated a brown precipitate.
Purification with 6% Nickel-IDA Agarose Ten bed volumes of Binding buffer (50mM NaH2Po4, 300mM NaCl, 10 mM imidazole, Urea 8M, pH:7) were added and mixed thoroughly at 4ºc overnight on a rotator. Having removed supernatant, E. coli lysate was added to gel and incubated at 4ºc overnight. Binding buffer was added 10 bed volumes and mixed gently on a rotatory shaker. The washing step (50mM NaH2Po4, 300mM NaCl, 20 mM imidazole, Urea 8M, pH:7) was repeated 4 times and 1.5 % Tween 20 was added to further reduce contamination. Suspensions were centrifuged at 500g for 5 minutes to sediment agarose. One bed volume of elution buffer (50mM NaH2Po4, 300mM NaCl, 250 mM imidazole, Urea 8M, pH=7) was added and mixed gently on a rotatory shaker. This suspension was centrifuged at 500g for 5 minutes and his-taged protein containing supernatant was transferred into new tube and stored on ice. After that, imidazole was removed by dialysis.
Post-translational modification was performed by Tyrosinase (from mushroom). To study adhesion, purified recombinant unmodified proteins of Mgfp-5-GvpA, GvpA-Mfp-3 and copolymer have been resolved in 5% acetic acid to halt auto-oxidation of DOPA residues and 25 mM ascorbic acid-containing buffer and have been modified with 10-unit mushroom tyrosinase to create DOPA at room temperature for 6h at pH=6.5 (17). BSA (18) and Cell-Tak were selected for negative and positive control respectively.
Self-assembly of Mfp-5-GvpA and GvpA-Mfp-3: After purification of Mfp-5-GvpA and Gvp-A-Mfp-3, protein concentration was measured by the bicinchoninic acid (BCA) assay. In phosphate solutions (pH=7), these proteins (45µM) were dissolved in. Then, pH was changed to pH=5. Following that, these proteins were stored overnight at 4ºc. Two chimeric proteins were incubated with a variety of molar ratios.
Nitroblue tetrazolium (NBT) used for staining Dopa-containing proteins: Acid-urea PAGE was performed to validate the presence of DOPA residues with redox cycling The nitrocellulose membranes were used for spotting 50 μg/mL bio-inspired adhesive samples.
After that, membranes with associated proteins were saturated in 0.0025 (m/v%) Congo red solution for 1 hour at RT. After washing with water, these membranes were incubated overnight.

Adhesion Force Measurement:
Adhesion force between our chimeric adhesion proteins on mica surface and colloidal probe (Silica) was measured by Atomic Force Spectroscopy (AFS) (21). AFM force measurements were