Stable, Long-Term Bacterial Production of Soluble, Dimeric, Disulfide-Bonded Protein Pharmaceuticals without Antibiotic Selection

Abstract

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide‐bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide‐bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high‐level expression of soluble, disulfide‐bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long‐term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.