[Expression, purification and characterization of rat procarboxypeptidase B in Pichia pastoris].
Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. In order to express the rat proCPB in Pichia pastoris, total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with Xho I and EcoR I , the fragment was inserted into pPIC9, and the recombinant plasmid was named as pPIC9-proCPB. By digestion with Sac I , the lined pPIC9-proCPB was transformed into Pichia pastoris strains GS115 with PEG1000 and integrated into their genomes. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris, and could be secreted into the supernatant in the culture. After optimizing the fermentation conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY (pH6.0) at 28CC, with addition of 0.5% casein. The yield of recombinant protein reached 500mg/L, achieving over 94% of total protein in the culture supernatant. The purity of recombinant CPB can reach 96% after two step phenyl sepharose F F purification, and 38% of total protein can obtained after optimizing the pufication method. Comparing to the specific activity 180u/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110u/mg. Mass spectrometry analyses showed the mass of the recombinant CPB was 35.1 kD, which is very close to the theory value 35.2 kD. Amino acid sequencing of N-terminal of recombinant CPB further indicated proCPB was expressed successfully and modificated correctly after translation.
Wang
De-Jie
DJ
College of Life Sciences, Jiangxi Sci- Tech Normal University, Nanchang 330013, China.
Miao
Lin
L
Chen
Hong
H
Li
Yan-Ying
YY
Chen
Hui-Peng
HP
Fang
Hong-Qing
HQ
D000818:Animals; D043424:Carboxypeptidase B / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013552:Swine
D011994:Recombinant Proteins; D043424:Carboxypeptidase B