title: A novel method for increasing production of mature proteins in the periplasm of Escherichia coli.
abstract: A novel strategy to obtain high-level production of mature proteins exported to the periplasm of Escherichia coli is described. It is based on a modified signal sequence generated by insertion of a coding sequence of the polypeptide precursor of interest at the BamHI site of the commercial vector pQE-30 resulting in an addition of a dodeca-peptide (MRGSH6GS) at the N-terminus 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 optimization of translation initiation and screening of host stains. The usefulness of this method is illustrated by overexpression of DsbC and DsbA. Induced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to comprise 20-30% of the total cellular proteins, and more than 95% of the expressed proteins were correctly processed and exported into the periplasm with yields of more than 100 mg per liter culture.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
authors.forename: X Q
authors.initials: XQ
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: X M
authors.initials: XM
authors.lastname: Pan
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D021382:Protein Sorting Signals / Q000737:chemistry; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D021382:Protein Sorting Signals