Cloning and expression of human colon mast cell carboxypeptidase.
OBJECTIVE
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.


CONCLUSIONS
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.
Chen
Zhang-Quan
ZQ
Allergy and Inflammation Research Institute, Medical College, Shantou University, Shantou 515031, Guangdong Province, China.
He
Shao-Heng
SH
D000595:Amino Acid Sequence; D001483:Base Sequence; D043422:Carboxypeptidases A / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology*; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data
D018076:DNA, Complementary; C468648:CPA3 protein, human; D043422:Carboxypeptidases A