A secretory system for bacterial production of high‐profile protein targets

Abstract

Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.

Accurate de novo design of hyperstable constrained peptides

“…Protein expression from E. coli was carried out using a large N-terminal fusion domain consisting of: the native E. coli protein OsmY to direct periplasmic and extracellular localization 42 , a deca-histidine tag for protein purification, and the SUMO protein Smt3 from Saccharomyces cerevisiae to chaperone folding and provide a mechanism for scarless cleavage of the fusion from the designed protein 43 . Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM.…”

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

“…Efforts in enhancing an ability to secrete proteins to growth medium are significant to enhance its soluble expression [6][7][8]. The secretion of recombinant pro teins into the extracellular space by E. coli presents advantages like easier purification and protection from proteolytic degradation.…”

High yield expression of proteins in <i>E. coli</i> for NMR studies

“…Recent studies propose the Outer membrane protein A (OmpA), Outer membrane protein F (OmpF) and Osmotically inducible protein Y (OsmY) as fusion partners secreting protein into the growth medium of E. coli depending on culture conditions via periplasm secretion. This is owing to the fact that the fusion partners are membrane proteins and there by render the purification procedure simple [26]. Recently, ELP (Elastin-like polypeptide) tags capable of self-cleavage and non-chromatographic protein purification in high cell density E. Coli fermentation has been reported [14].…”