Screening of genetic parameters for soluble protein expression in Escherichia coli.
Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets.
The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. erik.vernet@cpr.ku.dk
Erik
E
Vernet
Alexander
A
Kotzsch
Bjørn
B
Voldborg
Michael
M
Sundström
D003001:Cloning, Molecular; D003062:Codon; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007378:Interleukins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D012995:Solubility; D020349:Surface Plasmon Resonance
D003062:Codon; D004220:Disulfides; D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007378:Interleukins; D011993:Recombinant Fusion Proteins