Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing protein
(English)Manuscript (preprint) (Other academic)
Escherichia coli has a limited capacity to overexpress integral membrane proteins to amounts needed for structural studies. This is usually attributed to the limited capacity of the Sec transport machinery, shortage of accessory chaperons, sub-optimal codon usage, potentially “wrong” lipids, and lack of membrane space for the new proteins. A foreign, monotopic lipid glycosyltransferase was recently shown to induce the formation of extensive amounts of intracellular vesicles in E. coli. We show here that such vesicles can improve the expressed levels up to 3-4 times for a substantial fraction of integral membrane proteins tested. These had 2 to 12 transmembrane helices, and all had a C-terminally fused GFP reporter. Strongly overexpressed proteins yielded intensely green vesicles, of slightly lower buoyant density than the inner membranes. Most proteins could be detected in the vesicles. Multivariate sequence analyses indicated a correlation between sequence property features and expression levels, and factors analyzed involved protein mass, transmembrane segments, inside/outside loops, etc. It is concluded that this vesicular system can yield substantial improvements in expression levels, by creation of extra membranes and lateral space in E. coli.
IdentifiersURN: urn:nbn:se:su:diva-29069OAI: oai:DiVA.org:su-29069DiVA: diva2:229031