大鼠ACAT的N末端片段(氨基酸1～126)在大肠杆菌中的融合表达及纯化

Fusion expression and purification of rat ACAT (amino acid 1~126) in E.coli

目的： 重组法制备纯化乙酰辅酶Ａ：胆固醇酰基转移酶（ＡＣＡＴ）Ｎ末端膜外结构域（氨基酸１～１２６）。 方法：经ＰＣＲ扩增得到的ＡＣＡＴ的Ｎ末端膜外结构域（氨基酸１～１２６）用ＥｃｏＲＩ酶解，插入到表达载体ｐＧＥＸ－２ＴＫ中，得到重组表达质粒ｐＧＥＸ－２ＴＫ／ＡＣＡＴ（氨基酸１～１２６）。阳性重组子在大肠杆菌中经ＩＰＴＧ诱导表达ＧＳＴ－Ａ－ＣＡＴ（氨基酸１～１２６），重组表达菌裂解上清液经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ ＣＬ－４Ｂ亲和柱纯化。 结果：ＳＤＳ－ ＰＡＧＥ 和Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 分析显示得到了纯的ＧＳＴ－ＡＣＡＴ（氨基酸１～１２６）融合蛋白。 结论：ＡＣＡＴ的Ｎ末端膜外结构域（氨基酸１～１２６）的分离纯化为抗ＧＳＴ－ＡＣＡＴ（Ｎ 末端片段）抗体的制备及其ＡＣＡＴ结构和功能关系的进一步研究打下了基础。

Objectives: To isolate and purify GST fusion protein of the extra membrane domain of rat ACAT(amino acid 1~126). Methods: Rat ACAT (amino acid 1~126) amplified by PCR was digested with EcoRI and then subcloned into the expression vector pGEX 2TK to get recombinant plasmid pGEX 2TK/rat ACAT (amino acid 1~126). Upon IPTG induction , GST rat ACAT(amino acid 1~126)was expressed in E.coli .The expressed product was purified with Glutathione Sepharose CL 4B affinity chromatography from cellular lysate. Results:SDS PAGE and Western blotting analysis showed that purified GST fusion protein of rat ACAT(amino acid 1~126) was obtained. Conclusions:The purification of GST fusion protein of the extra membrane domain of rat ACAT(amino acid 1~126) may be useful in the production of antibody against N terminus of rat ACAT and in the studies of the relationship between the structure of ACAT and its functions.