Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B.
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.
Su-Xia
Li
L
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Yu-Jian
Zhang
Z
Li-Ping
Tian
T
Qin-Sheng
Yuan
Y
Yi
Gong
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043424:Carboxypeptidase B / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D014357:Trypsin
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D043424:Carboxypeptidase B; D014357:Trypsin