Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T, Yokoyama S
Cellular Signaling Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, Japan.
FEBS Lett. 1999 Jan 8;442(1):15-9. doi: 10.1016/s0014-5793(98)01620-2.
We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-isotope labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method.
我们提高了大肠杆菌无细胞蛋白质合成系统的生产率。首先，使用磷酸肌酸和肌酸激酶作为能量源再生系统，并对反应混合物的其他成分进行了优化。其次，通过对聚乙二醇溶液进行透析浓缩大肠杆菌S30细胞提取物，以提高合成速率。第三，在蛋白质合成过程中，将反应混合物对低分子量底物溶液进行透析，以延长反应时间。因此，氯霉素乙酰转移酶的产量提高到了6毫克/毫升反应混合物。通过该方法实现了用13C/15N标记的氨基酸对蛋白质进行稳定同位素标记以用于核磁共振光谱分析。
