Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection

Numerous biopharmaceuticals and other recombinant biotechnology products ar e made in prokaryotic hosts. However, bacterial production of native, biolo gically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of sol uble, native disulfide-bonded dimeric proteins in the Escherichia coli cyto plasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically acti ve proteins with up to six disulfide bonds have been produced with our expr ession system (Garces et al., 1997). However, plasmid instability during in duction limited its usefulness. We now report the stable, high-level expres sion of soluble, disulfide-bonded human uteroglobin without antibiotic sele ction. 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 induci ble after long-term continuous culture in nonselective liquid medium (at le ast 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive pr oteins for biochemical studies and biopharmaceutical/industrial purposes. A s a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than entero bacteria.