A novel recombinant chimeric bio-adhesive protein consisting of mussel foot protein 3, 5, gas vesicle protein A, and CsgA curli protein expressed in Pichia pastoris

Abstract

Despite various efforts to produce potent recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression levels. Here, a feasible expression system has been developed to produce strong recombinant fusion bioinspired protein using mussel foot protein 3 and 5 (Mfps) along with gas vesicle protein A (GvpA) of Anabaena flos-aquae, and a curli protein CsgA from E. coli, expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in yeast P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting, and their remaining dihydroxyphenylalanine (DOPA) was measured in the modified proteins by NBT assay. We further elucidated the mechanistic properties of obtained adhesive protein assembly in various pH levels based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that both combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher DOPA residues specially in acidic condition and tetrad complex which is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted from the fusion of GvpA and CsgA proteins with DOPA-containing Mfp proteins, expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications but also provides novel biological clues used for high-performance bioinspired biomaterial designation. Graphical Abstract

Biomimicry and 3D-Printing of Mussel Adhesive Proteins for Regeneration of the Periodontium—A Review

“…Moreover, the surrounding pH environment also plays an influential role in the adhesive nature of MAPs as, in a neutral or basic pH, DOPA can readily oxidize [ 33 ]. Despite these challenges, successful attempts have been made to produce functional and economical mussel-inspired protein recombinants, which mimic the adhesive characteristics of the natural MAPs found in mussels, that can be commercially produced [ 35 , 36 , 37 ]. One example of a successful attempt is a study by Bolghari and colleagues that investigated a novel combinational bio-adhesive chimeric protein that included the fusion of the gas vesicle protein A (GvpA) of Dolichospermum fosaquae , prokaryotic curli protein CsgA of Escherichia coli ( E. coli ), and DOPA-containing mussel foot proteins 3 and 5 (Mfp3, Mfp5), all expressed in methylotrophic yeast, P. pastoris [ 37 ].…”

“…Despite these challenges, successful attempts have been made to produce functional and economical mussel-inspired protein recombinants, which mimic the adhesive characteristics of the natural MAPs found in mussels, that can be commercially produced [ 35 , 36 , 37 ]. One example of a successful attempt is a study by Bolghari and colleagues that investigated a novel combinational bio-adhesive chimeric protein that included the fusion of the gas vesicle protein A (GvpA) of Dolichospermum fosaquae , prokaryotic curli protein CsgA of Escherichia coli ( E. coli ), and DOPA-containing mussel foot proteins 3 and 5 (Mfp3, Mfp5), all expressed in methylotrophic yeast, P. pastoris [ 37 ]. The authors concluded that P. pastoris a suitable expression system of adhesive proteins, allowing for a higher production rate and improved post-translational modifications for the function of the protein, in addition to the greater cohesion of proteins in acidic pH 3–5.…”

Bio-Based Adhesives for Orthopedic Applications: Sources, Preparation, Characterization, Challenges, and Future Perspectives

“…The binding and solidifying properties of MFP obtained from catechol side chain of DOPA are cross-linked with the surface of the substrate by chemical reactions. The MFP inspired the development of novel bio-based adhesives materials to fulfill the demands of satisfactory adhesive ability to wet surfaces by utilizing the advantages provided by adhesive proteins [11][12][13].…”

“…Because of the development that was carried out, a wide variety of tissue bio-based adhesives and hydrogels are now manufactured. However, their potential use as bone bio-based adhesives has been the subject of intensive research [13,47,[52][53][54][55][56].…”

In silico prediction and in vitro validation of the effect of pH on adhesive behaviour of the fused CsgA-MFP3 protein

“…We have recently achieved recombinant fused proteinous adhesive materials consist of mussel foot proteins, MFP3, MFP5, and GvpA and CsgA in both prokaryotic ( E. coli ) and eukaryotic expression systems (Irapour et al 2021 ; Bolghari et al 2022 ). In spite of inspiring properties and obtaining chimeric structure of obtained chimeric proteins, some obstacles are still needed to be solved prior to introduce a straightforward feasible approach for their industrial production.…”