A Novel Vector for Expression/Secretion of Properly Folded Eukaryotic Proteins: a Comparative Study on Cytoplasmic and Periplasmic Expression of Human Epidermal Growth Factor in E. coli

Expression of eukaryotic proteins in E. coli often results in their aggregation. Proper folding and solubility of therapeutical proteins are the pre-requisite for their bioactivity. This is not achieved in cytoplasmic expression in E. coli because of the absence of disulfide bonds formation. A novel expression/secretion vector was constructed which exploited β-lactamase signal sequence to translocate processed and soluble proteins into the periplasm of cells. Secretion of model proteins, β-lactamase and human Epidermal Growth Factor (hEGF) in M15/pSB and M15/pSE systems respectively, was confirmed by SDS-PAGE analysis and bioactivity assay. Secreted hEGF was found to be identical to authentic protein, in size, N-terminal amino acid sequence, biological activity and Western-blotting. The radioimmunoassay revealed 10-fold-higher level of hEGF expression in M15/pSE secretion system compared to that of cytoplasmic expression of the protein. The properly processed and in vivo folded hEGF demonstrated high solubility and bioactivity. The data obtained, evidenced in favor of M15/pSE expression system, which might be suitable to produce the small eukaryotic disulfide-bonded proteins for therapeutical applications or structural studies