毕赤酵母表达的HBV全长Pres蛋白的分离纯化

巴斯德-毕赤酵母工程菌株GS115-PreS经发酵在甲醇诱导下可高效表达分泌型全长PreS蛋白。Western blot证明发酵液中存在着可溶性的分子量为48kD的PreS蛋白和蛋白质颗粒，蛋白质颗粒主要成分为48kD的全长Pres蛋白和28kD的S蛋白，电镜观察发现蛋白质颗粒直径为30nm。发酵液经过脱盐、浓缩处理后，上清液经DEAESFF阴离子交换柱得到纯化的PreS蛋白；超速离心和蔗糖密度梯度离心得到蛋白颗粒。该颗粒的主要组分为全长PreS蛋白（PreS1＋PreS2＋S），还有少量的主蛋白（S）。ELISA检测证明全长PreS蛋白和蛋白颗粒有着良好的抗原性， P/N显示蛋白颗粒的抗原性比PreS蛋白的抗原性高。

The Purification of HBV Full-length PreS Protein in Pichia pastoris

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1+PreS2+S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.