Expression Of ProCarboxypeptidase B And A Snake Venom Metalloproteinase Alfimeprase In Pichia Pastoris

Naturally occurring rat carboxypeptidase B is produced from a precursor protein, preprocarboxypeptidase B, containing a 108 amino acid long N-terminal extension, which included the signal sequence (13 amino acid) and an activation peptide (95 amino acid). During transport of preprocarboxypeptidase B to the endoplasmatic reticulum, the signal peptide is cleaved off; the resulting enzymatically inactive proCPB precursor is secreted from the cell. The active CPB is then formed by cleavage of the activation peptide by trypsin in small intestine.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.CPB also can obtained by the engineering methods except for purifying from pancreas.Hratman et al and Li et al published the expression and purification of rat proCPB in Escherichia Coli respectively.Eaton et al and Ventura et al cloned and expressed human plasma proCPB and porcine proCPB in the methylotrophic yeast Pichia pastoris.Wang D J et al published the expression and purification of SD rat proCPB in the methylotrophic yeast Pichia pastoris using the AOX1 promoter.We amplify the GAP gene promoter from plasmid pIB2,construction of constitutive expression vector and the pGAP controlled constitutive expression of procarboxypeptidase B in Pichia pastoris in order to evaluate its possibility as an alternative to the AOX1 promoter. In this experiment, the sense and anti-sense primes were designed according to the synthesized gene sequence of the GAP promoter. Sac I and BamH I enzyme sites were cloned in the 5' and 3' terminals.The GAP gene of promoter was amplified by PCR from plasmid pIB2 and used to replace the AOX1 promoter(pAOX1) on plasmid pPIC9K-proCPB . A recombinant plasmid pGAPK-proCPB was constructed. It was linearized with the enzyme BglⅡand transformed into the Pichia pastoris GS115 competent cell by electroporation, and spread on MD plate. Positive clones were screened by HIS4 selectable marker and PCR. The genetic engineered strain pGAPK-proCPB/GS115 was cultured in a medium with a final concentration of 0.2% glucose. Recombinant proCPB was successfully expressed in Pichia pastoris for the first time,and could be secreted into the culture supernatant .The recombinant proCPB was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Phenyl FF. Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct-acting fibrin(ogen)olytic enzyme, but it is not hemorrhagic. The molecular weight of fibrolase is approximately 23,000 and the protein contains 203 amino acids in known sequence,with an N-terminal of the cyclized glutamine isoform formation.Alfimeprase is a mutant of Fibrolase at N-terminal sequence,contains 201 amino acids with an N-terminal sequence of SFPQR---,in contrast to the N-terminal sequence of fibrolase ,which begins with EQRFPQR---.The amino terminal modification in Alfimeprase solved the problem of the cyclized glutamine isoform formation.Alfimeprase can also promote thrombolysis by activation of the endogenous fibrinolytic system as well as Fibrolase. Alfimeprase has been demonstrated the proteolytic activity against fibrinogen .In this experiment, we try to express this metalloproteinase in the Pichia pastoris expression system, the sense and anti-sense primes were designed according to the synthesized gene sequence of the Alfimeprase. The gene of interest was amplified by PCR. Xho I and EcoR I enzyme sites were cloned in the 5' and 3' terminals. PCR production was connected to pPIC9 expression vector with T4 DNA ligase. A recombinant plasmid pPIC9-ALF was constructed. It was linearized with the enzyme BglⅡand transformed into the Pichia pastoris GS115 competent cell by electroporation, and spread on MD plate. Positive clones were screened by HIS4 selectable marker and PCR.The genetic engineered strain pPIC9-ALF/GS115 was induced with a final concentration of 1% methanol. Alfimeprase was expressed.In the preparation of the polyclonal antibody of Alfimeprase,the strain pET22b-ALF/BL21(DE3) genetically engineered was cultivated in L FML cultivation medium containing appropriate concentrate of ampicillin. It was induced withα-Lactose , and Alfimeprase was expressed. The collected E.coli cells were lysed using ultrasonic, and the aggregates collected by low-speed centrifugation. Inclusion bodies were washed and solubilized with strong protein denaturants, e.g. 6 mol/L urea. The solubilized inclusion bodies were mixed and emulsified with Freund's adjuvant. The solution was used to immunize BalB/C rats for three times. The last twice immunizations were strength. To get the serum of BalB/C rats, blood was obtained from tail and eyes and centrifuged at a low speed. The titer of the polyclonal antibody of Alfimeprase was identified by Elisa.