Expression And Purification Of Resilin-R5Fusion Protein And Characterization Of Its Material Properties

Resilin-R5fusion protein is an artificial chimera macromolecule that combinated with the natural super flexible protein Resilin and the silicon deposition peptide R5. Resilin is a natural biological protein with super elasticity often found in some insect exoskeleton, flight systems and sound systems. The elasticity and resilience variability of the crosslinked resilins are much higher than ordinary rubbers. The excellent mechanical properties of resilin may enable its applications in materials science, medicine, tissue engineering and other fields. R5peptide is a short19-amino-acid functional unit of the silaffin-1precursor polypeptide from Cylindrotheca fusiformis. R5peptide is capable of inducing and regulating the deposition of silica in Cylindrotheca fusiformis. In the silica deposition experiments of using different silica precursor, a different morphology of arched structure and fiber structure were obtained. The function of R5peptide in silica deposition in vitro suggests the R5peptide has potential applications in the fields of nanomaterials.A fusion protein Resilin-R5which was expected to have a superiority of high elasticity and silicon deposition was designed and expressed in Escherichia coli. At the same time, a facile purification method was built to obtain the new fusion protein. First, the recombinant resilin-r5gene, which constructed by fusing the diatoms R5peptide gene sequences to the3’end of Drosophila melanogaster resilin gene sequences, was synthesized after a rare codon optimization. Then, the new gene was inserted into the prokaryotic expression vector pET28a. The expression vector was transformed into E. coli BL21(DE3)pLysS strain, and the transformant was cultured in an auto-induced medium to get resilin-r5gene expressed. At the same time, the resilin-r5gene was also inserted into pUC57and the recombinant vector was transformed into E.coli AclpX, E. coli BW25113and E.coli DH5a. In order to obtain a high protein production, different media were used to culture the expression strains. At last, the His-tagged recombinant fusion protein was purified by’salting-out and heating’method and Ni ion column affinity chromatography. The expression efficient of the fusion Resilin-R5reached120mg per liter culture. And the full-length target protein Resilin-R5was confirmed by6×His-tag Western bloting and C-terminal protein sequencing of the obtained fusion protein.Further more, a series of Resilin-R5/chitosan composite films were prepared by blending with the Resilin-R5and the positive natural polysaccharide—chitosan at a certain ratio. And the swelling properties, permeability, water absorption, water retention, photocatalytic crosslinking properties, mechanical properties and silica deposition of the composite film material were tested. And the complex interaction between groups within the films, blend morphology and film performance were tested by Fourier translation infrared spectroscopy (FTIR), differential scanning calorimetry analysis (DSC) and microscopic observation. The results show that, swelling ratio of composite films showed a gradual downward trend with the increasing proportion of Resilin-R5and the lowest swelling ratio was approximately50%of the pure chitosan film. The permeability of the composite films were reached403g/m2·24h and392g/m2·24h when the Resilin-R5rates were at7%and33%respectively. The permeability was significantly higher than that of pure chitosan film. The water absorption and water retention of composite films were increased with the increasing of Resilin-R5proportion, reached to the highest of264%and231%, respectively, and was significantly higher than the pure chitosan film. The morphology of composite films showed multiple microsphere aggregation. The crosslinked composite films can emit blue fluorescence under UV. And the composite films can catalyze the hydrolysis of tetramethyl orthosilicate and form the particles sediment on the films surface or inside. The enlongation at break and the tensile strength at break of the composite films had reached41.6%and32.1MPa, when Resilin-R5ratio is13%, that significantly higher than the pure chitosan. The result of Fourier translation infrared spectroscopy and differential scanning calorimetry analysis showed the interaction between chitosan and Resilin-R5and a good blend performance showed between Resilin-R5and chitosan when Resilin-R5ratio is less than20%.In summary, in this paper resilin-r5fusion gene was designed and synthesized, and an efficient expression and purification method was achieved. At last, a new Resilin-R5protein biomaterials was obtained. The excellent material properties of Resilin-R5/chitosan composite film may enable its potential applications in medical dressings and sutures.