McLoughlin Sean Yu, Jackson Colin, Liu Jian-Wei, Ollis David
Research School of Chemistry, Building 35 Science Road, Australian National University, Canberra ACT 0200, Australia.
Protein Expr Purif. 2005 Jun;41(2):433-40. doi: 10.1016/j.pep.2005.01.012.
We devised a growth-based strategy for screening phosphotriesterase mutant libraries for variants with enhanced activity towards organophosphates that generate dimethyl phosphate when hydrolysed. Phosphotriesterase mutants were screened for activity by growing transformed Escherichia coli on agar plates containing methyl paraoxon as a sole phosphorus source. E. coli is capable of growth under these conditions when coexpressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) and the glycerophosphodiester phosphodiesterase from Enterobacter aerogenes (GpdQ). The latter enzyme can hydrolyse the dimethyl phosphate produced by the phosphotriesterase to methyl phosphate, which can then be used by E. coli as a source of phosphate. Phosphotriesterase was expressed from the lac promoter at levels such that its activity was growth-rate limiting. Cultures of the largest colonies (1% of the transformants) were assayed for activity towards paraoxon spectrophotometrically in microtitre plates. This process produced E. coli variants with higher whole cell activity than wild-type, which was found to be a consequence of increased protein expression rather than any increase in enzymatic activity. The mutations present in these mutant enzymes with increased expression were exclusively in the coding region, suggesting the improvement occurs post-transcriptionally.
我们设计了一种基于生长的策略，用于筛选磷酸三酯酶突变体文库，以寻找对水解时产生磷酸二甲酯的有机磷酸酯具有增强活性的变体。通过在含有甲基对氧磷作为唯一磷源的琼脂平板上培养转化的大肠杆菌，来筛选磷酸三酯酶突变体的活性。当共表达来自放射土壤杆菌P230的磷酸三酯酶（OpdA）和产气肠杆菌的甘油磷酸二酯磷酸二酯酶（GpdQ）时，大肠杆菌能够在这些条件下生长。后一种酶可以将磷酸三酯酶产生的磷酸二甲酯水解为磷酸甲酯，然后大肠杆菌可以将其用作磷源。磷酸三酯酶由lac启动子表达，其表达水平使其活性成为生长速率限制因素。对最大菌落（占转化体的1%）的培养物在微量滴定板中进行分光光度法测定对氧磷的活性。该过程产生了全细胞活性高于野生型的大肠杆菌变体，发现这是蛋白质表达增加而非酶活性增加的结果。这些表达增加的突变酶中存在的突变仅在编码区，表明这种改善发生在转录后。
