毕赤酵母分泌表达SAMS及表达产物活性分析Secreted Expression and Biological Assay of SAMS in Pichia pastoris
王莲哲;张现青;李洋;杨广笑;何光源;
摘要(Abstract)：
[目的]利用毕赤酵母分泌表达S-腺苷甲硫氨酸合成酶(SAMS)。[方法]以酿酒酵母(Saccharomyce cerevisiae)基因组DNA为模板,通过PCR扩增出S-腺苷甲硫氨酸合成酶基因2(sam2),构建重组毕赤酵母(Pichia pastoris)表达载体pPIC9Ks-am2,转化毕赤酵母GS115,以甲醇诱导表达分泌蛋白SAM合成酶,并用HPLC测定重组蛋白的酶活力。[结果]经SDS-PAGE鉴定重组蛋白分子量约50kD,比理论值42.5 kD稍大,可能是分泌过程中糖基化等加工造成。体外酶促反应结果显示,伴随甲醇诱导及初步纯化过程的进行,蛋白活力逐步提高,纯化后比活力为61.48 U/mg。[结论]该研究首次实现了SAM合成酶的胞外表达,为开发和建立体外酶促法生产SAM工艺奠定了基础。
关键词(KeyWords)： SAM;毕赤酵母;pPIC9K;分泌表达
基金项目(Foundation): 国家“973”计划项目(2002CB111302)
作者(Author): 王莲哲;张现青;李洋;杨广笑;何光源;
Email:
DOI: 10.13989/j.cnki.0517-6611.2009.10.005
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