Hirel P H, Lévêque F, Mellot P, Dardel F, Panvert M, Mechulam Y, Fayat G
Laboratoire de Biochimie, CNRS UA240, Palaiseau, France.
Biochimie. 1988 Jun;70(6):773-82. doi: 10.1016/0300-9084(88)90107-1.
The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.
本文描述了一系列携带大肠杆菌甲硫氨酰 - tRNA合成酶基因（metG）衍生物的质粒构建。这些质粒可实现该酶天然形式或截短形式的表达，并便于产物的纯化。为便于对催化活性极低的修饰酶进行表征，构建了一种特殊载体，其中metG与β - 半乳糖苷酶基因（lacZ）读框融合。该质粒表达甲硫氨酰 - tRNA合成酶 - β - 半乳糖苷酶嵌合蛋白，已证明其具有两种酶的活性。这种杂种蛋白可通过一步β - 半乳糖苷酶亲和层析进行纯化。甲硫氨酰 - tRNA合成酶部分可通过温和的蛋白酶解再生，从而提供了一种纯化和研究突变蛋白的简单方法。
