Preparation Of Recombinant Kex2 In Pichia Pastoris And Its Enzyme Activity Identification

Kex2 endopeptidase,also called Kexin,Paired-basic endopeptidase,Prohormone-processing endoprotease,these names reflects the enzymatic properties of Kex2 from different angles.Kex2 gene from Saccharomyces cerevisia,which belong to subtilisin family,is a calcium-dependent serine proteolytic enzyme.Kex2 recognize the double basic amino acid residues(Lys-Arg,Arg-Arg)and Pro-Arg of protein or polypeptide specifically,and cut the peptide bond of the second carboxy-terminal amino acid residues(R-).As a prototype of precursor processing enzyme,Kex2 contributed greatly to the research progress of a variety of other eukaryotic proprotein.In addition,it also found that Kex2 able to complete the enzyme processing of some prohormone precursors and protein precursor in vivo and in vitro environment.Therefore,due to the specificity of its restriction sites,Kex2 has shown its position in the biopharmaceutical field gradually in recent years.To further study,we must get a lot of Kex2 enzyme to carry out various studies in vitro and in vivo.At present,the studies for Kex2 structure and activity have been more comprehensive,for Kex2 recombinant constructs and preparation is still very few.Althoμgh there have been reported in the literature,but to built the high expression of recombinant engineering bacteria for fermentation and complete the subsequent purification process and get Kex2 pure product has been relatively little,paper is devoted to the completion of the study objectives and content.Objectives: To design nucleotide sequences and protein sequence of recombinant Kex2,and construct the recombinant yeast strain to correctly express and enhance Kex2 enzyme expression.To explore the fermentation processing,which is the foundation for industrial production.To establish the stable,reproducible,high recovery rate of purification process,and we want to obtain pure Kex2.To establish the methods of measuring the concentration and activity of Kex2,then it was used for digestion experiments proteins of the substrates to verify its activity.Methods:(1)Design recombinant Kex2 cDNA sequences Refer to the literature,select Kex2 protein sequence 2-660 residues and design the N-terminal His-tag LEKRSARGSHHHHHH to facilitate downstream purification;design the cDNA of Kex2 according to codon preference of P.Pastoris,design stop codon TGA and TAA,design restriction sites XhoI(CTCGAG)and NotI(GCGGCCGC).(2)Construct recombinant expression strain pPICZαA-Kex2 / X-33 Synthesise the whole cDNA sequence,obtain a glycerol bacteria containing the target sequence.After extraction plasmids,the vector and target gene were double-digested by Xho I and Not Iand then ligated,transform into Top10F’,then screening recombinant plasmid pPICZαA-Kex2.With Sac I linearized recombinant plasmid was transformed into the X-33,positive clones were induced by methanol,using method such as resistance to flat screen high expression strain,identified and determined to use it for fermentation.(3)Establish the fermentation process Establish the shake flask fermentation of recombinant bacteria to screen the optimum temperature,pH,dissolved oxygen and other conditions in the initial stage,then grope the fermentation by ferment tank,determine the fermentation medium,induction time,induction of mode and so on.(4)Establish purification process of fermentation supernatant According to the innovative design of the protein sequence,preferably try Ni metal chelation column as the first step of purification.Then,select ion exchage chromatography to remove excess impurities.(5)Active identification of homemade Kex2 samples Due to enzyme specificity of Kex2,we choose three different substrates include living substrates,peptides and proteins,to establish their digestion methods and to verify enzyme activity and specificity of homemade Kex2.Results:This study successfully constructed pPICZαA-Kex2/X-33 recombinant bacteria,completed the recombinant expression of Kex2 in Pichia pastoris system;selected out the highest expression of the strain and completed the 10 L fermentation tank fermentation;Then innovatively purified by the Ni ion chelate column firstly and then ion exchage chromatography after desalting column to get recombinant Kex2 sample whose electrophoresis purity is more than 95%;Then the concentration and purity was tested,the most important is the efficiency and specificity determination of the enzyme by three substrate,proved that homemade Kex2 sample can play its role in digestion when the substrate respectively was Boc-QRR-pNA,short peptides(containing KR recognition site),protein level(glargine precursor,comprising KR and RR recognition sites),and no wrong cut.This has great significance for the future applications and industrial production of Kex2.