Heterologous Expression of Bovine Prochymosin in Pichia pastoris GS115

Objectives: In present research we evaluate the expression of this critical enzyme in a eukaryotic system for future use in cheese industry. Materials and Methods: We have cloned bovine prochymosin gene in methylotrophic yeast, P. pastoris, using pPIC9K as an expression vector. The recombinant plasmid was transformed into the host by electroporation, and it was expressed in optimum con‌ditions (temperature 29oC, 200 rpm, 2% methanol for induction, and 5 days of incuba‌tion). Transcription and expression of the recombinant prochymosin was evaluated by the reverse transcription polymerase chain reaction (RT-PCR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis as well as western blotting and enzyme-linked immunosorbent assay (ELISA). Results: In optimum conditions, only a low level of this heterologous protein was de‌tected using ELISA method and subsequently confirmed by RT-PCR. Conclusions: Since it has been reported that P. pastoris is an appropriate host for the ex‌pression of recombinant proteins, a low level of expression of prochymosin in this host should be explored in our future research.