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: Haibo Wang, Duoduo Yuan, Rong Xu, Cheng-Wu Chi
• Journal: Acta biochimica et biophysica Sinica (Acta Biochim Biophys Sin (Shanghai)) Vol. 43 Issue 7 Pg. 568-75 (Jul 2011) ISSN: 1745-7270 [Electronic] China
• PMID: 21659382 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Complementary
• Recombinant Proteins
• chymotrypsinogen C
• Chymotrypsinogen
• Serine Endopeptidases
• Chymotrypsin
• chymotrypsin C
• Metalloendopeptidases
• peptidyl-Lys metalloendopeptidase

Topics

• Amino Acid Sequence
• Animals
• Chymotrypsin (genetics, isolation & purification)
• Chymotrypsinogen (chemistry, isolation & purification)
• Cloning, Molecular
• DNA, Complementary (genetics)
• Escherichia coli (genetics)
• Inclusion Bodies (genetics)
• Metalloendopeptidases (metabolism)
• Molecular Sequence Data
• Pancreas (enzymology)
• Recombinant Proteins (chemistry, isolation & purification)
• Serine Endopeptidases (chemistry, isolation & purification)
• Swine