The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis.
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.
Listwan
Pawel
P
Bioscience Division, MS-M888, Los Alamos National Laboratory, Bikini Atoll Rd, SM30, Los Alamos, NM 87545, USA. plistwan@lanl.gov
Pédelacq
Jean-Denis
JD
Lockard
Meghan
M
Bell
Carolyn
C
Terwilliger
Thomas C
TC
Waldo
Geoffrey S
GS
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups)
D011506:Proteins; D049452:Green Fluorescent Proteins; D048630:Polyketide Synthases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase