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

Despite various efforts to produce strong 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 level. Here, a feasible expression system has been developed to produce strong recombinant fusion protein using Mfp3 and Mfp5 along with two curli proteins, GvpA and CsgA expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting and its remaining dopa were measured in the modified proteins by NBT assay. We further elucidated mechanistic properties of obtained adhesive protein assembly in various pH based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher dopa residues majorly in acidic condition and tetrad complex that is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted through 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 using for high-performance bioinspired biomaterials designation.