基于rhaSR嵌合操纵子的构建及其在大肠杆菌中表达的研究

通过PCR等重组DNA技术,构建了含rhaSR启动子表达调控元件、RhaR基因、报告基因gst(谷胱甘肽-S-转移酶)的两个嵌合操纵子,并插入大肠杆菌表达载体pALEX中构成pALEX-PR1和pALEX-PR2。其中pALEX-PR2的RhaR基因上游为原有的SD序列,而pALEX-PR1的RhaR基因上游则插入了增强的SD序列。把这两个重组表达质粒分别转入大肠杆菌BL21(DE3)中,报告基因gst能够在L-鼠李糖诱导下表达,其表达量是非诱导条件下的4～5倍,且pALEX-PR1的表达量是pALEX-PR2的3.14倍。以上结果表明,gst的表达既受L-鼠李糖诱导,同时又受RhaR的正调控。SDS-PAGE结果显示,GST占大肠杆菌培养物总可溶蛋白的5.41%(W/W),平均1L培养物可获得3.0mg纯化的GST。酶活性分析表明,所构建的嵌合操纵子表达的GST保持了正确的构型且具有很高的活性。

Chimeric Operons Based on rhaSR and Their Expression in Escherichia coli

Using gene recombinant techniques,two chimeric operons containing rhaSR promoter,RhaR gene,and reporter gene gst(glutathione S-transferase)were constructed,and each was inserted into the E.coli expression vector pALEX to form pALEX-PR1 and pALEX-PR2.The pALEX-PR2 contained a native SD sequence in the upstream region of rhaR,while the pALEX-PR1 contained an enhanced SD sequence.Two plasmids were then transformed into E.coli BL21(DE3).The reporter gene gst within both chimeric operons expressed 4 to 5 folds higher with L-rhamnose induction than without the induction.Under the induction of L-rhamnose,the GST expression of the pALEX-PR1 was 3.14 folds than the pALEX-PR2.Our results suggested that the expression of gst was positively regulated by the induction of L-rhamnose and the RhaR expressed from the same chimeric operon.Furthermore,SDS-PAGE results showed that GST accounted for about 5.41%(W/W)of the total soluble proteins of the E.coli culture.An average of 3.0 mg purified GST was obtained from 1 L culture.The results of enzyme activity analysis showed that the GST expressed by reporter gene gst of our chimeric operons kept the correct configuration and high activity.