Miles Luke A, Crespi Gabriela A N, Han Sen, Hill Andrew F, Parker Michael W
St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
Methods Mol Biol. 2011;752:17-28. doi: 10.1007/978-1-60327-223-0_2.
In vitro translation systems derived from a wide range of organisms have been described in the literature and are widely used in biomedical research laboratories. Perhaps the most robust and efficient of these cell-free systems is that derived from Escherichia coli. Over the past decade or so, experimental strategies have been developed which have enhanced the efficiency and stability of E. coli cell-free systems such that we can now prepare recombinant proteins on a scale suitable for purification and analysis by biophysical and structural biology techniques, which commonly require relatively large quantities of protein. This chapter describes in detail the protocols employed in our laboratory to prepare translationally active E. coli extracts and to synthesise proteins on a milligram scale from these extracts.
文献中已描述了源自多种生物体的体外翻译系统，这些系统在生物医学研究实验室中被广泛使用。其中最强大且高效的无细胞系统或许是源自大肠杆菌的系统。在过去十年左右的时间里，已开发出一些实验策略，这些策略提高了大肠杆菌无细胞系统的效率和稳定性，以至于我们现在能够以适合通过生物物理和结构生物学技术进行纯化和分析的规模制备重组蛋白，而这些技术通常需要相对大量的蛋白质。本章详细描述了我们实验室用于制备具有翻译活性的大肠杆菌提取物以及从这些提取物中毫克级合成蛋白质的方案。
