Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B

• Published in: Protein and peptide letters Vol. 10; no. 6; p. 581
• Main Authors: Su-Xia, Li, Yu-Jian, Zhang, Li-Ping, Tian, Qin-Sheng, Yuan, Yi, Gong
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2003
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Carboxypeptidase B - biosynthesis; Carboxypeptidase B - genetics; Carboxypeptidase B - isolation & purification; Cloning, Molecular; Enzyme Activation; Enzyme Precursors - biosynthesis; Enzyme Precursors - genetics; Enzyme Precursors - isolation & purification; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Molecular Sequence Data; Pancreas - enzymology; Rats; Rats, Sprague-Dawley; Recombinant Proteins - isolation & purification; Substrate Specificity; Trypsin
• ISSN: 0929-8665
• DOI: 10.2174/0929866033478627

A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved. Further research showed that the obtained recombinant carboxypeptidase B could substitute carboxypeptidase B isolated from pancreas.