以大肠埃希氏菌肽脱甲酰基酶为靶点的高通量筛选模型的建立(英文)  

Establishment of a high throughput screening model targeted on peptide deformylase (PDF) of E.coli

作　　者：唐先兵[1] 司书毅[1] 张月琴[1] 

机构地区：[1]中国医学医学院中国协和医科大学医药生物技术研究所,北京100050

出　　处：《中国抗生素杂志》2003年第10期621-626,共6页Chinese Journal of Antibiotics

摘　　要：本文用 PCR法从大肠埃希氏菌 K-12中克隆出肽脱甲酰基酶 (peptide deformylase,PDF)基因 ,通过测序确证与文献报道的 pdf基因序列完全一致。将 p df连接到原核蛋白表达载体 p ET-3 0 -a(+ )上 ,并转化到大肠埃希氏菌 BL 2 1(DE3 )菌株中 ,IPTG诱导表达。过量表达的 PDF酶用 Q Sepharose HP离子交换柱和 Superdex 75纯化得到高纯度 Ni-PDF。应用荧光测试仪Polar Fluostar检测 PDF对底物 For-Met-Ala-Ser的水解活性 ,PDF酶的已知抑制剂放线酰胺素 (actinonin)为阳性对照 ,通过优化酶反应条件 ,建立了以The pdf gene from E.coli K-12 was a mplified by PCR. The amplified DNA sequence was conformed to be identical to pdf of E.coli reported by sequencing. The pdf was subcloned to the protein expression vector pET-30a(+) and was transformed to host cell, E.coli BL21(DE3). The overexpression of PDF was induced with 1mmol/L isopropyl-1-t hio-β-D-galactopyranoside(IPTG). Ni-PDFS were purified on a Q-Sepharose HP ion exchange column in the presence of NiCl 2 and further purified by the size exclusion chromatography using Superdex75. The activity of PDF was assayed with the fluorescence detector PolarFluostar, selecting the for-Met-Ala-Ser f or the reaction substrate of Ni-PDFs enzyme. By optimizing the conditions of en zyme reaction and using actinonin, a known inhibitor of PDF, as the positive con trol. A high throughput screen model targeted to Ni-PDFs was established.

关 键 词：肽脱甲酰基酶 高通量筛选模型 放线酰胺素 

分 类 号：R965.1[医药卫生—药理学]