具有生物活性的人促肾上腺皮质激素释放激素原在大肠杆菌中的表达：特性与纯化

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification.

Castro M G, Spruce B A, Savva D, Lowry P J

Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, England.

Int J Biochem. 1990;22(11):1341-9. doi: 10.1016/0020-711x(90)90318-w.

Abstract

Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

摘要

人促肾上腺皮质激素释放激素原（CRH）在大肠杆菌TG2菌株中作为与β-半乳糖苷酶的融合蛋白表达。2. 经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和考马斯亮蓝染色后，在携带重组质粒pre-proCRH(10 - 196)[ph PPC(10 - 196)]的TG2细胞裂解物中鉴定出一条与β-半乳糖苷酶pre-proCRH融合蛋白相对应的140 kDa条带。通过蛋白质印迹法和双位点免疫放射分析确认了融合蛋白的身份。3. 通过在8 M尿素存在下的离子交换色谱法从分离、洗涤和溶解的包涵体中纯化融合蛋白。4. 在摩尔基础上比较促肾上腺皮质激素释放活性时，嵌合CRH前体的效力为合成的r/h CRH(1 - 41)的4%。