Heterologous Expression?Purification Of The Human Carboxypeptidas B And Its Enzyme Activity Study

Carboxypeptidase B(CPB)is a kind of metalloproteinase,which can hydrolyze protein or peptide substrate.At present,CPB is used in the enzyme digestion of recombinant insulin and other products as an important tool enzyme.In recent years,The expression system of Pichia pastoris is rapid,widely used,mature in high-density fermentation technology,simple in production process,and conducive to large-scale production.Therefore,this study attempts to construct a recombinant human CPB zymogen Pichia pastoris expression strain and establish a relatively systematic fermentation and purification production process,in order to obtain high purity and activity recombinant human CPB finally.In this study,the PCPB target protein gene fragment obtained by digestion is ligated with the digested pPIC9 vector to construct the recombinant plasmid pPIC9-PCPB.Then the recombinant plasmid pPIC9-PCPB is transferred into Pichia pastoris expression strain GS115 to construct Pichia pastoris expression strain of recombinant Human PCPB.The constructed strain is activated and transferred to BMGY flask medium,culturing at 30? and 200 rpm shaking speed,the final volume of 1%methanol is added every 24h to induce,and the recombinant human CPB zymogen expression strain with the highest expression was screened after inducing for 72 hours.Optimization of the seed medium:Tryptone-20g/L,Yeast extract-10g/L,Glycerin-10g/L,Potassium dihydrogen phosphate-1.2g/L,Disodium hydrogen phosphate dodecahydrate-2.2g/L,Defoamer-25?l/L.After culturing for 24 hours at a temperature of 30? and a rotational speed of 220rpm,the bacterial OD600 of the recombinant human CPB zymogen can reach 10?20.In the 19L fermenter,Optimization of the fermentation process parameters of recombinant human CPB zymogen:the inoculation amount of the seed in the fermenter is 1.5%,the induced temperature is 25?,the induced pH is 6.0,the induced OD600 is about 200,the optimum methanol feeding rate is increasing 1.0 ml/Lh in every three hours.After culturing for 116?120 hours,the recombinant human CPB zymogen expression level is above 800mg/L.Use the IMAC affinity chromatography and the Qff ion exchange for purification of recombinant human CPB zymogen.Optimization of the purification process parameters of recombinant human CPB zymogen:Keep the pH of the recombinant human CPB zymogen sample unchanged before loading and select 20mM Tris+0.3M NaCl system as the IMAC affinity chromatography buffer system;Select 10mM/20mM Tris+5mM NaCl as the Qff ion exchange chromatography buffer system.The recombinant human CPB purity reaches 95%,more than domestic similar product reports(90%),and the recombinant human CPB enzyme activity reaches more than 120U/mg.At the production scale of 500L fermenter,further enlargement according to the optimized fermentation and purification process,the recombinant human CPB enzyme expression is stable and reaches more than 760mg/L,the purity of recombinant human CPB can reach more than 95%,and the activity of the enzyme can reach above 200 U/mg.The recombinant human CPB production process established in this study is simple,reproducible and stable.The recombinant human CPB enzyme can be applied to the trial production of insulin,and the enzyme digestion yield can reach more than 35%.