Backbone H(N), N, Calpha, C' and Cbeta assignments of the 19 kDa DHFR/NADPH complex at 9 degrees C and pH 7.6.
Backbone H(N), N, Calpha, C' and Cbeta assignments of the 19 kDa DHFR/NADPH complex at 9 degrees C and pH 7.6.
复制标题
19 kDa DHFR/NADPH 复合物在 9 摄氏度和 pH 7.6 下的主链 H(N)、N、Calpha、C 和 Cbeta 分配。
DOI：
10.1023/a:1008330429173
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发表时间：
2000
影响因子：
2.7
通讯作者：
Wright,PE
中科院分区：
文献类型：
--
作者：
Zaborowski,E;Chung,J;Kroon,G;Dyson,HJ;Wright,PE
Methods and resultsE. coli BL21 (DE3) competent cells were transformed with a pET-22b plasmid containing the E. coli K-12 strain wild type DHFR gene. Cells were grown at 37◦ C in minimal medium containing (15N) ammonium sulfate and (13C) glucose as the only source of nitrogen and carbon, and overexpression was induced by IPTG when cells reached OD600= 1. The enzyme was purified from the soluble fraction using a variation of the protocol of Falzone et al.(1994). A 2D 15N-HSQC experiment was used to monitor sample stability and to optimize solution conditions for NMR experiments. Probe head temperature was calibrated using a neat methanol sample and controlled to within±0.1◦ C. We used enzymatically reduced NADPH (Sigma), following reports (Rafter and Colowick, 1957) that it is less acid labile than the chemically reduced material. The highest pH at which there was no major intensity loss of the NMR signal (due to amide-proton/solvent exchange) was 7.6. At this pH, and with a phosphate buffer concentration of
