Listwan Pawel, Pédelacq Jean-Denis, Lockard Meghan, Bell Carolyn, Terwilliger Thomas C, Waldo Geoffrey S
Bioscience Division, MS-M888, Los Alamos National Laboratory, Bikini Atoll Rd, SM30, Los Alamos, NM 87545, USA.
J Struct Funct Genomics. 2010 Mar;11(1):41-9. doi: 10.1007/s10969-009-9077-8. Epub 2010 Jan 13.
Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse were well correlated with sonication. Two other methods, Bugbuster and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability.
在大肠杆菌中生产蛋白质包括在培养物中进行高水平表达，随后收获细胞，最后将细胞破碎或裂解以释放表达的蛋白质。我们使用机器人平台和我们实验室开发的方法，将三种高通量化学裂解方法与超声处理进行比较[1]。在相同的表达条件下，所有裂解方法释放的可溶性蛋白质程度各不相同。对于一组96种测试蛋白质，我们使用我们的分裂绿色荧光蛋白来定量裂解后可溶性和不溶性蛋白质部分。使用SoluLyse裂解后，可溶性蛋白质的量和在可溶性部分中回收的百分比与超声处理都有很好的相关性。另外两种方法，即Bugbuster和溶菌酶，与超声处理的相关性不佳。当需要精确测量蛋白质溶解度时，例如在测试佐剂、生长培养基、温度时，或者在确定截短或序列变异对蛋白质稳定性的影响时，考虑裂解方法对蛋白质溶解度的影响尤为重要。
