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.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2003年第10期621-626,共6页
Chinese Journal of Antibiotics
