Cloning and expression of human colon mast cell carboxypeptidase.
AIM: To clone and express the human colon mast cell carboxypeptidase (MC-CP) gene. METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to construct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E. coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P. pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid sequencing and enzyme assay. RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E. coli was purified with amylose affinity chromatography and heparin agarose chromatography. SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine. CONCLUSION: The cDNA encoding human colon mast cell carboxypeptidase can be successfully cloned and expressed in E. coli and P. pastoris, which will contribute greatly to the functional study on hMC-CP.[1]References
- Cloning and expression of human colon mast cell carboxypeptidase. Chen, Z.Q., He, S.H. World J. Gastroenterol. (2004) [Pubmed]
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