Cloning And Expression Of Gene Encoding Carboxypeptidase Inhibitor From Potato

Potato carboxypeptidase inhibitor (CPI) is a small, 39-residue, globular protein that specially inhibits carboxypeptidase A and carboxypeptidase B from human plasma in vivo, which is also named thrombin-activatable fibrinolysis inhibitor (TAFI). It was reported that CPI is likely to become a potential assisted thrombolytic drug, which can overcome the obstacles of restrictive dose and easy hemorrhage in clinical treatment of thrombus with the traditional thrombolytic drugs. At present, CPI is mostly purified from potato, the technical process of which is more complex and the recovery rate is lower, and the poisonous solanine can not be removed completely. Molina etc successfully constructed a recombinant Escherichia coli which can expressed excretively, however, the purification of reCPI from culture liquid should suffer 4 steps at least, and the yield is only 52% ultimately.The gene cpi encoding potato CPI was amplified by polymerase chain reaction, and cloned into PGEX-4T-1 vector to construct the expression plasmid pGCPI. pGCPI was sequenced and was transformed to E. coliBL21. E. coliBL21(pGCPI) could express soluble reCPI-GST at 37℃with 0.2 mmol/L IPTG as inducer.The conditions for E. coli BL21(pGCPI) cell growth and expression in the LB medium in shake flask were optimized. The optimized condition is that the concentration of IPTG is 0.2mmol/L; the induction begins at A600 reaches 0.5⁰.7 and last for 5 hours at 30℃; the inoculation size is 5%. The optimized medium suitable for high density fermentation is composed of: glucose 10 g/L, (NH4)2SO4 1 g/L, peptone 3 g/L, citric acid 2 g/L, KH2PO4 15 g/L, MgSO₄·7H₂O 1.5 g/L, trace elements solution (TES) 5 mL/L. The effect of dissolved oxygen (DO) concentration and the feeding of carbon and nitrogen source for the growth of E. coli BL21(pGCPI) were studied at a 3.7L fermentor. When the DO was controlled above 30%, carbon and nitrogen source was feeded at log phase,the dry cell weight and the expression level of reCPI-GST reached 26.2 g/L and 24.8% of the total cell protein, respectively.The reCPI was successfully separated and purificated through four convenient steps: glutathione Sepharose FF affinity chromatography, cleavage with thrombin, glutathione Sepharose FF affinity chromatography and Centrifugal Filter. About 698μg reCPI was obtained from the cell harvested from 500mL cultural liquid, the relative purity and the recovery of reCPI were 98.9% and 60.5%, respectively. There was no evident difference between reCPI and natural CPI in carboxypeptidase A or carboxypeptidase B inhibitory effect on enhancing fibrinolysis in vitro.