Expression of Snow Flea (Hypogastrura harveyi) Antifreeze Protein HhAFP in Pichia pastoris and Its Antifreeze Effect
ZHONG Wen-Qian1, YAN Qian-Qian1, HU Rui-Qin1, CHEN Liang-Biao1,2*
1 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; 2 Center for Aquacultural Breeding Research/Key Laboratory of Exploration and Utilization of Aquatic Resources, Ministry of Education/International Research Center for Marine Biological Sciences, Shanghai Ocean University, Shanghai 201306, China
Abstract：Antifreeze proteins (AFPs) are a type of macromolecular proteins that can bind to the surface of ice crystals to inhibit the growth of ice crystals. Pichia pastoris is a system that can express foreign proteins. It can use methanol as a carbon source to express the target protein. In order to obtain high-yield active antifreeze protein, the insect snow flea (Hypogastrura harveyi) antifreeze protein (HhAFP) was selected. XhoⅠ and XbaⅠ restriction sites were added to the 5' end and 3' end of the target gene through PCR. 6×His tag was added to the 3' end of the target gene that could be used for identification and screening. The target gene was added to the expression vector pPICZαA suitable for the P. pastoris expression system through transformation, and the recombinant expression vector pPICZαA-HhAFP was successfully constructed. pPICZαA-HhAFP was transformed into P.pastoris expression strain X-33 by electroporation, and cultured in a 29 ℃ incubator until positive yeast transformants grew. Two positive yeast transformants were then screened out using bleomycin (zeocin) and methanol. The expression of these 2 yeast strains was induced and cultured for 72 h using BMM medium containing 1% methanol at 29 ℃, 250 r/min, and pH 6.0. The yeast supernatant was purified using affinity chromatography, and the obtained purified product was dialyzed and concentrated. It was analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF) to verify that the protein was the recombinant protein HhAFP. The recombinant protein HhAFP was added to the cell cryopreservation solution to test its antifreeze activity. The results proved that HhAFP could significantly improve the survival rate of cells after cryopreservation and reduce cell damage. By further optimizing the expression conditions of HhAFP, the culture temperature was determined to be 28 ℃ and the methanol concentration was 1.5%. The recombinant protein HhAFP obtained after 72 h of expression and culture could reach 527 mg/L. This study provides a technical approach for the high-yield expression of active antifreeze proteins by P. pastoris and the industrialization of antifreeze proteins.
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