Expression, Purification And Characterization Of Carboxypeptidase B In Pichia Pastoris

Carboxypeptidase B(CPB) (EC 3.4.17.2) is a metalloenzyme, which contains one zinc atom per molecule and selectively hydrolyzes Arginine and Lysine from the carboxyl terminus of protein or peptides. CPB has been widely used for commercial and research purposes now, such as in protein sequence analysis, C-terminal trimming of proteins and peptides, and in the diagnosis of acute pancreatitis. In the activation of proinsulin to insulin, CPB was one of the two necessary enzymes, trypsin and CPB. However, commercially available CPB purified from porcine pancreas was very expensive, and was not totally free of other proteases, such as trypsin and chymotrypsin.This thesis was purposed to product a high-activity and cheaper recombinant CPB by genetic engineering method. In this work, the coding sequence of the procarboxypeptidase B (proCPB) gene was obtained from SD rat fresh pancreas by RT-PCR, and the codes of proCPB gene 5'region were optimized to yeast bias codes. The optimized coding sequence of proCPB was inserted into the pPIC9 vector and transformed into Pichia pastoris GS115 strain. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris for the first time, and could be secreted into the culture supernatant. After optimizing the expression conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY(pH 6.0), at 28℃, with addition of 0.5%(m/v) casein acid, 3 mmol/L PMSF, and 0.5%(v/v) methanol per 12 h. The yield of recombinant protein reached 500 mg/L, and achieved over 94% of total protein in the culture supernatant. The recombinant proCPB was subjected to trypsin enzymatic cleavage to produce active CPB, and CPB was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Phenyl FF, as a result, 50 mg CPB per litre cell culture was achieved. Comparing to the specific activity 180 U/mg of standard CPB purchased from Sigma, the specific activity of recombinant CPB was 110 U/mg. The stability analysis of recombinant CPB suggested that the CPB was very stable and a slight hydrolysis was occured even it was stored for 5 months at 4℃.