Chávez Adán, García-Huante Yolanda, Ruiz Beatriz, Langley Elizabeth, Rodríguez-Sanoja Romina, Sanchez Sergio
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico DF, Mexico.
J Ind Microbiol Biotechnol. 2009 May;36(5):649-54. doi: 10.1007/s10295-009-0533-z. Epub 2009 Feb 11.
It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (Dog(R)) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His(6)) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. coelicolor mutant lacking the sco2127 gene (Deltasco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.
已知对2-脱氧葡萄糖具有抗性的天蓝色链霉菌变种卡氏变种（Dog(R)）突变体表现出葡萄糖转运缺陷、低葡萄糖激酶（Glk）活性以及对碳分解代谢物阻遏（CCR）不敏感。这种表型可以由来自天蓝色链霉菌的编码SCO2127的576bp基因多效性互补，这表明该蛋白质参与了CCR过程。在本研究中，将sco2127区域亚克隆到pQE30中，并使其转录产物（SCO2127-His(6)）过表达。该过程允许纯化SCO2127（使用镍琼脂糖树脂）并产生多克隆抗体。在蛋白质印迹分析中，这些抗体对来自天蓝色链霉菌和卡氏链霉菌变种的蛋白质提取物产生阳性反应，表现为一条34 kDa的单条带。使用来自缺乏sco2127基因的天蓝色链霉菌突变体（Deltasco2127）的提取物未检测到蛋白质。与其可能参与CCR过程一致，在补充有50和100 mM葡萄糖的基本培养基中生长的天蓝色链霉菌的对数生长期检测到SCO2127。此外，当利用50 mM葡萄糖时，在微生物生长的后期SCO2127和残余葡萄糖浓度同时下降。
