Tuning Escherichia coli for membrane protein overexpression.
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
Wagner
Samuel
S
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Klepsch
Mirjam M
MM
Schlegel
Susan
S
Appel
Ansgar
A
Draheim
Roger
R
Tarry
Michael
M
Högbom
Martin
M
van Wijk
Klaas J
KJ
Slotboom
Dirk J
DJ
Persson
Jan O
JO
de Gier
Jan-Willem
JW
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D050576:Membrane Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D020543:Proteome / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D029968:Escherichia coli Proteins; D050576:Membrane Fusion Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020543:Proteome; D014764:Viral Proteins; C412443:YIDC protein, E coli; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
J Mol Biol. 1986 May 5;189(1):113-30
3537305
Nat Methods. 2006 Apr;3(4):303-13
16554836
Annu Rev Biochem. 1993;62:749-95
8394684
J Mol Biol. 1996 Jul 19;260(3):289-98
8757792
J Mol Biol. 1991 May 5;219(1):37-44
2023259
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
10829079
Biotechniques. 2006 Mar;40(3):355-64
16568824
Biochim Biophys Acta. 2003 Feb 17;1610(1):3-10
12586374
Mol Microbiol. 2001 Apr;40(2):397-413
11309122
Trends Biotechnol. 2006 Aug;24(8):364-71
16820235
Proc Natl Acad Sci U S A. 1970 Jul;66(3):773-9
4913210
Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5722-7
18391190
J Biol Chem. 2006 Apr 14;281(15):10024-34
16352602
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12250-4
8618879
Protein Sci. 2005 Aug;14(8):2011-7
15987891
J Mol Biol. 1968 Dec;38(3):421-6
4887878
Methods Enzymol. 1990;185:60-89
2199796
Mol Cell Proteomics. 2007 Sep;6(9):1527-50
17446557
J Bacteriol. 1992 Jan;174(2):619-22
1729251