Engineering Protein Folding To Improve Heterologous Proteins Secretory Expression In Pichia Pastoris

Heterologous protein secretion in Pichia pastoris is often limited to relatively low levels.One bottleneck is that the evaluated heterologous protein may result in protein unfolding and/or misfolding and increase ER(endoplasmic reticulum)stress.To enhance protein folding capacity,the disulfide isomerase PDI,the ER chaperone protein BiP and the unfolded protein response transcription factor HAC1 was each co-expressed with different heterologous proteins in P.pastoris.Their effect on secretory expression of heterologous proteins was investigated with the model proteins yEGFP(yeast-enhanced green fluorescent protein),Gal(?-galactosidase),and CPC acylase(Cephalosporin C acylase).In this study,according to genome annotation information of P.pastoris GS115,six endogenous folding factors(PDI1,PDI2,PDI3,BiP1,BiP2 and HAC1)with the potential to promote protein secretion and expression were obtained.Among them,PDI2,PDI3 and BiP2 have not been reported previously.Co-expression of these folding factors had no significant impact on the growth of yEGFP recombinant strains.For the Gal recombinant strains,BiP1 co-expression led to the decrease in the maximum specific growth rate by 27%,and PDI1 co-expression resulted in an increase in the final cell concentration by 35%.The maximum specific growth rate of CPC acylase recombinant strains was decreased by 39%after BiP2 co-expression.Compared with the parental strain,co-expression of all folding factors improved the expression of CPC acylase,with the maximum increase up to 60%(PDI2 and BiP1),and the CPC acylase activity reached 173.9 DCW U/g(PDI2).Gal expression was decreased after co-expression of these folding factors.The most significant decrease of the Gal expression level was observed for BiP2,PDI2 and HAC1 to reach only 0.3%,1.1%and 0.7%of that in the parental strain,respectively.The expression of yEGFP was affected to different degrees.The highest expression level was obtained with the co-expression of HAC1(2-fold),while yEGFP expression was decreased with the co-expression of PDI2,PDI3 and BiP2.yEGFP is easy to be secreted,the effect of folding factors on the secretion of yEGFP was not notably.Both CPC acylase and Gal were difficult to be secreted.Co-expression of BiP2 or HAC1 increased CPC acylase secretion rate by more than 2-fold.Although the co-expression of folding factors increased the Gal secretion rate except PDI2,with the highest level of a 14.9-fold increase(BiP2),due to the decreased expression caused by co-expression of these factors,the maximum extracellular Gal yield only increased by 27%(BiP1).There was a correlation between the expression and transcription level of heterologous proteins,therefore the transcription level generally reflected the level of protein expression.Taken together,it could be concluded that BiP1 and HAC1 were better than rest folding factors for promoting secretory expression of heterologous proteins in P.pastoris.