非融合型重组人骨唾液蛋白在毕赤酵母中的高效表达及纯化

High Expression of Non-Fusion Recombinant Human Bone Sialoprotein in Pichia Pastoris and Its Purification

通过正交设计对毕赤酵母GS115／pPICZαAN-hbsp工程菌的发酵条件（菌体密度、甲醇诱导浓度、初始pH值、诱导时间）进行优化。采用重组人骨唾液蛋白（recombinant human bone sialoprotein，rhBSP）单克隆抗体与Aldehyde-Resin HP FF介质偶联制备免疫亲和层析柱，纯化非融合型rhBSP，并用Western blotting和补体抑制实验检测非融合型rhBSP的抗原性和活性。毕赤酵母GS115／pPICZαAN-hbsp工程菌最佳发酵优化为：菌体密度OD600达到45甲醇诱导剂量为1．5％，pH值6．0，诱导时间2d，最大表达量为0．126mg／ml。经免疫亲和层析法纯化后，SDS-PAGE显示非融合型rhBSP为43～66ku的分散条带，纯度在90％以上，Western-blotting显示rhBSP具有良好的抗原性，补体实验证明纯化的非融合型rhBSP活性很好。毕赤酵母GS115／pPICZαAN-hbsp工程菌发酵条件经优化后能高效表达非融合型rhBSP，应用免疫亲和层析方法纯化的非融合型rhBSP纯度好，活性高，为进一步研究非融合型rhBSP打下了基础。

The expression system of pichia pastoris GS115/pPICZαA-hbsp, including density of pichia pastoris, the concentration of methanol, pH, and induced time, were optimized by orthogonal design to increase the yield of rhBSP expressed in Pichia pastoris. The non-fusion rhBSP was purified by immune affinity chromatography column prepared by coupling rhBSP monoclonal antibody （McAb）with Aldehyde-Resin HP FF. Western blotting and complementary inhibitory analysis were used to detect the activity of non-fusion rhBSP. After optimizing the expression condition, a higher production could be obtained when GS115/pPICZαAN-hbsp was induced in BMMY for 2 days, with addition of 1.5% （V/V） methanol, OD600 being 45 and pH being 6.0. The yield of non-fusion rhBSP reached 0. 126 mg/ml. After purification with immune affinity chromatography, there was a broad band between 43446 kD of non- fusion rhBSP by SDS-PAGE detection. The purity of non-fusion rhBSP was 90%. Western-blotting showed that the purified rhBSP had antigenic activity and non-fusion rhBSP could inhibit MDA-MB- 231BR evasion of complement-mediated attack. Non-fusion rhBSP expressed in recombinant pichia pastoris with high yield, high purity and keeping natural activity provide, the molecular foundation for the further research.