Heterologous Expression of Bovine Prochymosin in <italic>Pichia pastoris</italic> GS115

Abstract

<em>Objectives:</em> In present research we evaluate the expression of this critical enzyme in a eukaryotic system for future use in cheese industry. <em>Materials and Methods:</em> We have cloned bovine prochymosin gene in methylotrophic yeast, <em>P. pastoris, </em>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). <em>Results:</em> In optimum conditions, only a low level of this heterologous protein was de‌tected using ELISA method and subsequently confirmed by RT-PCR. <em>Conclusions: </em>Since it has been reported that <em>P. pastoris </em>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.