Dale G E, Schönfeld H J, Langen H, Stieger M
Department of Biology, Pharmaceutical Research New Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Protein Eng. 1994 Jul;7(7):925-31. doi: 10.1093/protein/7.7.925.
The production of the trimethoprim-resistant type S1 dihydrofolate reductase (DHFR) from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES is described. The simultaneous overproduction of the chaperonins with DHFR results in an increased solubility of the enzyme. We compare the time course of production of active type S1 DHFR by measuring enzyme activity in cells overproducing or not overproducing the chaperonins. Although co-overproduction of the chaperonins reduces the total production level of type S1 DHFR, the amount of soluble and active DHFR is increased several-fold in comparison with cells producing only DHFR. Thus, the higher concentrations of GroES and GroEL in cells overproducing the chaperonins partially protect DHFR from aggregation, resulting in higher concentrations of soluble and active DHFR in the cell. Furthermore, we also demonstrate that the chaperonins can improve in vitro refolding yields of type S1 DHFR. These results suggest that it is possible to purify suitable amounts of trimethoprim-resistant type S1 DHFR for X-ray crystallographic studies.
描述了在过量表达伴侣蛋白GroEL和GroES的大肠杆菌细胞中，由金黄色葡萄球菌产生耐甲氧苄啶的S1型二氢叶酸还原酶（DHFR）。伴侣蛋白与DHFR同时过量表达会导致该酶的溶解度增加。我们通过测量过量表达或未过量表达伴侣蛋白的细胞中的酶活性，比较了活性S1型DHFR的产生时间进程。虽然伴侣蛋白的共同过量表达降低了S1型DHFR的总产生水平，但与仅产生DHFR的细胞相比，可溶性和活性DHFR的量增加了几倍。因此，过量表达伴侣蛋白的细胞中较高浓度的GroES和GroEL部分保护DHFR不发生聚集，导致细胞中可溶性和活性DHFR的浓度更高。此外，我们还证明伴侣蛋白可以提高S1型DHFR的体外重折叠产率。这些结果表明，有可能纯化出适量的耐甲氧苄啶的S1型DHFR用于X射线晶体学研究。
