title: Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas.
abstract: Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: Duoduo
authors.initials: D
authors.lastname: Yuan
authors.forename: Rong
authors.initials: R
authors.lastname: Xu
authors.forename: Cheng-Wu
authors.initials: CW
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000235:genetics* / Q000302:isolation & purification*; D002919:Chymotrypsinogen / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000235:genetics; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification; D013552:Swine
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; C096020:chymotrypsinogen C; D002919:Chymotrypsinogen; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C117569:chymotrypsin C; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase