人载脂蛋白基因apoAⅠ在毕赤氏酵母中的表达

Expression of Human Apolipoprotein AⅠ Gene in Yeast Pichia pastoris

将化学法合成的apoAⅠ基因插入分泌型载体 pPIC9K .将重组的 pPIC9K apoAⅠ用BglⅡ酶切后 ,转化PichiapastorisGS115菌株 ,筛选获得G4 18高抗性转化子 .转化子经摇瓶发酵和甲醇诱导 ,上清液用SDS PAGE检测 ,有明显的rApoAⅠ表达 .经Phenyl sepharose 6 (FastFlow)疏水层析柱和SephadexG 5 0 (coarse)分子筛层析 ,得到rApoAⅠ蛋白纯品 .经Westernblotting ,N末端氨基酸顺序测定证明 ,毕赤氏酵母表达的rApoAⅠ与人血浆提取的ApoAⅠ基本一致 .

ap oAⅠgene,which was chemically synthesized,was inserted into secretion vectors pPIC9 and pPIC9K. The recombined pPIC9K- apo AⅠ was digested by Bgl Ⅱ and transformed into Pichia pastoris strain GS115, then the transformants with high G418 resistance were selected. After the transformants grew in the shaking flask and was induced by methynol, the SDS-PAGE analysis of supernatant showed that they expressed rApoAⅠ in a high level. rApoAⅠwas isolated and purified by Phenyl-sepharose 6 (Fast Flow) and Sephadex G-50 (coarse) gel permeation chromatography. The results obtained through Western blotting and N-terminal amino acid sequence determination indicated that purified rApoAⅠprotein expressed by Pichia pastoris had very similar characteristics compared with those of natural ApoAⅠprotein purified from human serum.