High-level Expression And Characterization Of Pichia Pastoris Kex2

Kex2 protease is a precursor-processing protease from yeast.The Kex2 recognize the double basic amino acid residues and cut the peptide bond of the second carboxy-terminal amino acid residues.So it plays a key role in the yeast protein secretion pathway and widely used in eukaryotic cell research.Pichia pastoris expression system is widely used in expression of Kex2 protease,but the research on the activity and enzymatic characterication of Pichia pastoris Kex2 protease?PPKex2?has rarely been reported.In addition,the low expression level of Kex2 protease leads to high production cost.In our previous studies,PPKex2 was homologously expressed using Pichia pastoris expression system,and the enzymatic characterication of PPKex2 were studied and compared with SCKex2.Methods such as high-density expansion of culture,truncation of C-terminal sequences,promoter optimization and co-expression of chaperones were carried out to improve the PPKex2 expression level.The main works and innovative results are listed as follows:?1?Firstly,these kex2 genes were cloned from the genomes of Pichia pastoris and Saccharomyces cerevisiae,inserted into pPIC9K expression vector,and then transformed into Pichia pastoris GS115.The expression of PPKex2 and SCKex2 were induced by methanol and purified with anion exchange chromatography?Q-FF?.Finally,the enzymatic characteristics of these Kex2 protease were characterized.The protein concentration and enzyme activity of PPKex2 achieved the maximum of 0.42 mg·mL^-1 and 30.2 U·L^-1.The specific enzyme activity of PPKex2 in the supernatant was seven times higher than that of SCKex2.Enzymatic characterization showed that PPKex2 was similar to SCKex2 and had the optimal activity at pH 8.0-9.0 and 37oC.In terms of stability,PPKex2 was most stable at pH 7.0.PPKex2 was more stable than SCKex2 in alkaline pH and less stable than SCKex2 in acidic pH.Meanwhile,the thermostability of PPKex2 is lower compared with that of SCKex2.The kinetic analysis showed that the k_cat and k_cat/K_m of PPKex2 was 4.8-and 3.3-fold higher than that of SCKex2,respectively.?2?The PPKex2 expression strain was studied by high-density fed-batch fermentation.The results indicited that in a 3 L fermentor,the protein concentration achieved the maximum 0.88mg·mL^-1,which was twice of the shake flask;the maximum activity reached at 38.6 U·L^-1,which was 20%higher than that in the shake flask.In order to improve the expression level,the fermentation condition was optimizied.In the methanol feed stage,when the mixed carbon source with methanol-sorbitol mass ratio of 30:1 was added,the exogenous expression of PPKex2 was increased by 16%.And when the 1%casein amino acid was added,the exogenous expression of PPKex2 was increased by 9%.?3?The effects of the promoters of FDH1,FLD1,DAS1,DAS2,THI4,THI11,HSP82 and GAP on the expression of PPKex2 were studied.The results showed that the expression of PPKex2 under the FDH1 promoter was the highest.The protein concentration and enzyme activity achieved the maximum of 1.03 mg·mL^-1 and 61.4 U·L^-1,which were 2.5-and 2-fold higher than the AOX1 promoter strain,respectively.In addition,the FDH1 and GAP promoters were selected to construct a double promoter co-expression strain,but the double promoter co-expression strain grew abnormally and the expression level was lower than the parent.?4?In addition,we tried to improve the expression by truncation of the C-terminal of PPKex2 and co-expression of seven chaperone?CPR6,ESS1,HSP82,KAR2,SSA1,YDJ1 and STI1?,but the results did not show any improvement of the expression level of PPKex2.