A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 (III) chain in Pichia pastoris

Abstract

Recombinant human full-length mature collagen α1 (III) chain (rhCOL3A1) was secreted by Pichia pastoris GS115, using the Saccharmyces cerevisiae a-mating factor prepro signal, and the theoretical molecular weight of rhCOL3A1 was 95.344 kDa. The gene cloned from human placenta, was designed and cloned into expression vector pPIC9K under the control of a strong inducible promoter AOX1.The expression stage of rhCOL3A1 was sensitive to different carbon ratios through mixed fermentation. LCMS/ MS analysis and western blotting demonstrated that the recombinant human full-length mature collagen a1 (III) gene was successfully expressed in P. pastoris GS115 during the methanol induction stage. Furthermore, an effective strategy of mixed fermentation was established to express rhCOL3A1 in shake flash. Compared to single carbon induction, when induced with mixed carbon at the ration of 0.8 (glycerol/methanol), the time corresponding to the highest yield of rhCOL3A1 (1.27 g/L) was drastically reduced by 50%. The same conclusion was observed from RT-qPCR. Consequently, a new strategy which was more time-saving and effective was provided for the large-scale producing the full-length mature rhCOL3A1.