大肠杆菌T蛋白的聚合状态分析

Polymerization Status Analysis of Escherichia coli T-protein

目的解析大肠杆菌T蛋白的四级结构。方法利用分段克隆法克隆表达了T蛋白及其两个独立结构域分支酸变位酶(CM,T/1-94)和预苯酸脱氢酶(PDH,T/93-373),再利用SDS-PAGE测得变性条件下T蛋白、CM和PDH的Mr分别为42×103,11×103和32×103,HPLC测得天然条件下CM和PDH的Mr分别为25×103和63×103,再用化学交联法分析聚合状态。结果显示T蛋白、独立结构域CM和PDH均为二聚体。结论说明大肠杆菌T蛋白二聚体是通过CM与CM、PDH与PDH相连的。

OBJECTIVE To describe the tertiary structure of T-protein from Escherichia coli .METHODS T-protein, CM and PDH domains were cloned and expressed separately. The Mr determined by SDS-PAGE under denatured condition were 42× 10^3, 32× 10^3 and 11× 10^3 respectively for T-protein, PDH domain and CM domain, which were identical to the theoretical Mr. The calculated Mr of HPLC under the native condition were 63× 10^3 and 25×10^3 respectively for PDH domain and CM domain. Chemical cross linking was employed to determine the polymerization status of T-protein,for its Mr was over-range in HPLC determination. RESULTS It is obvious that the Ms were doubled for PDH and CM domain under native condition. All of T-protein, CM and PDH are dimmers, Which was testified by both HPLC and chemical cross-linking experiments. CONCLUSION T-protein is a dimmer and it bonds together through adhesion of CM to CM and PDH to PDH domains.