Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli.
The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10x10(3) rel. mol. mass units (pheromaxein A) and 8x10(3) rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S.Tag and His.Tag coding regions being upstream of the inserted gene. Soluble pheromaxein A-thioredoxin (pheroA/trx) and pheromaxein C-thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.
Austin
Corrine
C
Unilever R&D Colworth, Colworth House, Sharnbrook, MK44 1LQ, Bedfordshire, UK. corrine.austin@unilever.com
D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002850:Chromatography, Gel; D017931:DNA Primers; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C054734:pheromaxein