A novel method for increasing production of mature proteins in the periplasm of Escherichia coli

A novel strategy to obtain high-level production of mature proteins exporte d to the periplasm of Escherichia coli is described. It is based on a modif ied signal sequence generated by insertion of a coding sequence of the poly peptide precursor of interest at the BamHI site of the commercial vector pQ E-30 resulting in an addition of a dodeca-peptide (MRGSH(6)GS) at the N-ter minus of the precursor. The modification does not affect correct processing of the modified signal nor proper folding of the target protein, resulting in an untagged native product. The method is simple for avoiding onerous o ptimization of translation initiation and screening of host stains. The use fulness of this method is illustrated by overexpression of DsbC and DsbA. I nduced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to compr ise 20-30% of the total cellular proteins, and more than 95% of the express ed proteins were correctly processed and exported into the periplasm with y ields of more than 100 mg per liter culture.