High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium

AbstractSecreted phospholipase A2s (sPLA2s) are a group of enzymes with 6–8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.Keywords: Basal salts mediumhuman sPLA2 group IIEPichia pastorispurity AcknowledgmentsWe thank the Research Centre for Life Sciences at the University of Science and Technology of China (Hefei Anhui) for the support in the mass spectrometry of samples.Disclosure statementNo potential conflict of interest was reported by the author(s).Additional informationFundingThis work was supported by the Postdoctoral Science Foundation of China [2021M702053]; the National Natural Science Foundation for Young Scientists of China [31900917]; and the Science and Technology Innovation Plan of Colleges and Universities of Education Department of Shanxi Province [2019L0423].