Koretsky A P, Traxler B A
Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA 15213.
FEBS Lett. 1989 Jan 16;243(1):8-12. doi: 10.1016/0014-5793(89)81206-2.
A cDNA encoding the B isozyme of creatine kinase (CKB) has been expressed in Escherichia coli from a fusion with lacZ carried by lambda gt11. Western blots indicate that a stable polypeptide with the appropriate mobility for the beta-galactosidase-creatine kinase (beta-gal-CKB) fusion protein cross-reacts with both beta-gal and CKB antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the lambda gt11-CKB construct have a CK activity of 1.54 +/- 0.07 mumol/min per mg protein. The fusion protein appears to provide this activity because immunoprecipitation of protein with beta-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli.
编码肌酸激酶B同工酶（CKB）的cDNA已在大肠杆菌中通过与λgt11携带的lacZ融合而表达。蛋白质免疫印迹表明，具有与β-半乳糖苷酶-肌酸激酶（β-gal-CKB）融合蛋白相适应迁移率的稳定多肽能与β-半乳糖苷酶和CKB抗血清发生交叉反应。在对照大肠杆菌中未检测到明显的CK活性；然而，含有λgt11-CKB构建体的细胞提取物具有的CK活性为每毫克蛋白质1.54±0.07微摩尔/分钟。融合蛋白似乎提供了这种活性，因为用β-半乳糖苷酶抗血清对蛋白质进行免疫沉淀会导致提取物中CK活性丧失。通过在添加了肌酸的培养基上生长的大肠杆菌的31P NMR谱中检测到磷酸肌酸（PCr）峰，证明了该酶在体内具有活性。与在哺乳动物脑和肌肉中一样，检测到的PCr峰对大肠杆菌的能量状态敏感。
