Repeat Sequences Like Collagen Expression Vector Construct And Secretion In Pichia Pastoris Expression

Collagen represents the chief structural protein accounting for approximately 30% of all vertebrate body protein, and plays a significant role in multiple biological functions, such as maintaining the structure of various tissue and effecting cell attachment and proliferation and tissue development. A human-like collagen monomeric gene (gel) was designed and synthesized according to human typeⅢcollagen, the unique character of collagenous domain, especially, which is made up of total repeat of "Gly-x-y" unit, and aimed at improving the water-solubility. Through series of gene cutting/ligation and transforming E. coli as a cloning host, the gel repeated in the same orientation forming six times ploygene, which was then inserted into the EcoRI/NotI sites of vector pPIC9K that contains a HIS4 selectable marker, by designed adaptors. After verifying the validity of construction by DNA sequencing, the recombinant vector pPIC9KG6 was electroporated into the Pichia pastors GS115, and the transformants were screened on YPD medium with gradient concentration of G418 to determine the copy number of the human-like collagen ploygene. The human-like collagen was expressed and secreted by methanol fed-batch inducement that added per 24 hours for maintaining at 0.5% concentration, using picked high-level expression strain. In order to analyze the extracellular proteins, SDS-PAGE was performed to the fermentation supernatant, the result of which showed the apparent molecular weight of the human-like collagen was about 112 kDa. Furthermore, some inducement factors were optimized, the orthogonal experiment came out that expression level has been increased to about 60.1mg/L from about 8.6mg/L, when fermentation was performed at 1.5%/24h methanol adding; 3.0OD₆₀₀/mL yeast concentration originally; 60h inducing time and pH 6.6 at the beginning. As one more step trying, the recombinant human-like collagen yield by GS115 was purified at ordinary methods, which can obtain crude product by 40%～60% saturation of ammonium sulfate precipitation, or 40%～80% acetone precipitation. All this work might provide a base level feasible beginning to achieve the scale-up production of recombinant human-like collagen, which has plentiful application.