The expression of bovine pancreatic procarboxypeptidase a in the pichia pastoris yeast

Objectives: This report aimed to obtain recombinant procarboxypeptidase A (rProCPA) from bovine pancreatic which could degrade ochratoxin A after tryptic activation. Methods: According to the Pichia pastoris preference codon usage, ProCPA gene was synthetized and plasmid pPIC9K/ProCPA was constructed. After linearization of pPIC9K/ProCPA vector with Sal I, the P. pastoris GS115 strain was transformed by electroporation. GS115 was induced to produce rProCPA in liquid BMMY medium. The cell-free supernatant was identified by SDS-PAGE (12.5%) and MALDI-TOF spectrometry. rProCPA was hydrolyzed with TPCK treated trypsin to produce rCPA, and 4 μg/mL of OTA was degraded by rCPA. The degradation rate was determined by high performance liquid chromatography (HPLC). Results: Bovine pancreatic ProCPA was expressed in the methylotrophic yeast P. pastoris GS115 and secreted into the culture medium by means of the α-mating factor signal sequence. SDS-PAGE analysis indicated a molecular weight of 47 ku protein was obtained and showed no difference with theoretical value. The result of mass spectrometric analysis demonstrated that the protein was Bovine pancreatic ProCPA. The expression quantity of the total protein was reached to 190 mg/mL after induction with methanol, and rProCPA was 150 mg/mL. rCPA could degrade OTA, the degradation rate was 72.3% of 4 μg/mL OTA by HPLC and degradation product OTα was produced. ©, 2015, Chinese Institute of Food Science and Technology. All right reserved.