Expression of actin in Escherichia coli. Aggregation, solubilization, and functional analysis.
Expression of actin in Escherichia coli. Aggregation, solubilization, and functional analysis.
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DOI：
10.1016/s0021-9258(18)38260-7
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发表时间：
1990-10
期刊：
影响因子：
--
通讯作者：
S. Frankel;J. Condeelis;L. Leinwand
中科院分区：
文献类型：
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作者：
S. Frankel;J. Condeelis;L. Leinwand
在大肠杆菌中表达了野生型盘状网柄菌肌动蛋白(42 KDa)和截短型肌动蛋白。氨基酸末端测序表明，截短的物种由两个肽组成，它们是Met-119和Met-123内翻译起始的结果。经超声波处理或法式压榨裂解后，所有肌动蛋白都以高度不容溶解的聚集体形式存在。当细菌直接裂解到Sarkosyl洗涤剂中时，大部分肌动蛋白是可溶的，去除Sarkosyl后，仍有50%以上的肌动蛋白是可溶的。用DNase I亲和层析和凝胶过滤纯化全长野生型肌动蛋白。该物种能够以ATP敏感的方式聚合和结合肌球蛋白，表明它是天然的。亲和层析表明，截短形式的肌动蛋白与DNase I结合的程度与真核生物合成的肌动蛋白相同，这表明该方法适用于突变形式的肌动蛋白。因此，利用Sarkosyl的裂解程序可能被证明在分离一些其他蛋白质方面是有用的，这些蛋白质通常是可溶的，但在细菌表达后变得不可溶。
Wild type Dictyostelium discoideum actin (42 kDa) and a truncated form of actin were expressed in Escherichia coli. Amino-terminal sequencing indicated that the truncated species was composed of two peptides, which were the result of internal translation initiation at Met-119 and Met-123. After sonication or French press lysis, all of the actin was present in highly insoluble aggregates. When bacteria were lysed directly into Sarkosyl detergent, most of the actin was soluble, and greater than 50% remained soluble after Sarkosyl was removed. Full-length wild type actin was purified using DNase I affinity chromatography and gel filtration. This species was able both to polymerize and to bind myosin in an ATP-sensitive manner, indicating it was native. Affinity chromatography demonstrated that the truncated form of actin bound DNase I to the same extent as actin synthesized in eukaryotes, indicating the applicability of this approach to mutant forms of actin. Thus, lysis procedures utilizing Sarkosyl may prove useful in isolating some of the other proteins which are normally soluble but become insoluble after bacterial expression.