Optimization Of Enterokinase Secretory Expression In Pichia Pastoris

Enterokinase is a type of serine proteases in the duodenum of mammals.People who born with lacking enterokinase would suffer from serious intestinal malabsorption,diarrhea and vomiting which eveulally affects body growth and development.Naturally,enterokinase consists of a light chain and a heavy chain,which are linked by a pair of intermolecular disulfide bonds.Studies have shown that the light chain expressed alone has biocatalytic activity,while the heavy chain plays a role in anchoring the light chain to the brush marginal membrane of the small intestine.Enterokinase specifically recognizes the DDDDK sequence and cleaves carboxyl terminal peptide bond of the lysine in the enzyme digestion reaction.The first amino acid at the N-terminal of the target protein is the same as that of the natural protein,and no excess amino acid residues are introduced.Owing to the characteristic of enterokinase,it has been widely used in the field of biomedicine as a tool enzyme.Currently,the production of enterokinase in recombinant Pichia pastoris is limited by the expression level,which hinders related industrial applications.To improve the secretion level of enterokinase and realize its large-scale production in near future,we have used six different signal peptide sequences?SP1,SP2,SP3,SP4,SP7 and SP8?to replace the amino acid sequence of?-factor signal peptide pre-region region.The effects of six signal peptides on the secretory expression of enterokinase in P.pastoris GS115 were studied.Compared with?-factor signal peptide,the signal peptide with SP1 sequence in the pre-region region significantly increased the secretory expression of enterokinase from 6.8 mg·L^-1 to 14.3 mg·L^-1.In addition,the activity of enterokinase was enhanced from 2390±212 U·m L^-1 to 4995±378 U·mL^-1.Based on optimizing the SP1 signal peptide,further steps have been conducted to further increase the secretory expression of enterokinase in P.pastoris GS115.In this case,endogenous proteins like Bi P,PDI,and Kex2 of P.pastoris GS115 were expressed.The co-expression of BiP had no effect on the enzyme activity,while the co-expression of PDI and Kex2 increased the enzyme activity of enterokinase to 6178±432 U·m L^-1 and 7219±489 U·m L^-1,respectively.However,PDI and Kex2 were co-expressed at the same time,and the enzymatic activity of enterokinase was only shown 5616±320 U·mL^-1.In the comparison of extracellular secretion efficiency,it was found that the co-expression of P.pastoris GS115 endogenous proteins PDI and Kex2 increased the specific enzyme activity.In the study of fusion expression,some proteins,such as Trx A,DsbA,DsbC,and SUMO,were fused at the N-terminal of enterokinase,while GSGSGSDDDDK sequence was added in the middle of the fusion protein and enterokinase for self-cleavage.The results showed that the activity of enterokinase decreased significantly in the fermentation supernatant when the N-terminal fusion of TrxA and DsbA,and enterokinase was inactive when the N-terminal fusion of DsbC and SUMO.The solvent accessibility of the fusion protein complex was analyzed,and it was found that the exposure of the DDDDK sequence to the protein surface did not affect the self-cleavage of enterokinase.Results showed that the N-terminal sequence has an important effect on the activity of enterokinase.In order to explore the effect of the N-terminal sequence on the activity and further optimize the secretory expression of enterokinase in P.pastoris GS115,three amino acid combinations of short peptides were randomly introduced before the initial sequence of enterokinase.Four mutant strains GP1EFM,GP1RNL,GP1LKR,and GP1WLR were obtained by 48-well plate screening.The enzyme activity could significantly increase by introducing the short peptide?WLR?at the N-terminal of enterokinase.The enzyme activity could be increased from 4995±378 U·mL^-1 to 15145±920 U·mL^-1 and the specific enzyme activity was1174600±53100 U·mg^-1.Based on the substrate kinetic analysis,the K_m increased and the substrate binding ability decreased after the introduction of short peptides into the N-terminal of enterokinase,but the substrate catalytic ability was improved.In summary,the researches lay a foundation for the related applications of enterokinase in the future.