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

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• 作者：Giuditta Mantile ; Chana Fuchs ; Eleonora Cordella-Miele ; Alessandro Peri ; Anil B. Mukherjee ; and Lucio Miele
• 刊名：Biotechnology Progress
• 出版年：2000
• 出版时间：February 2000
• 年：2000
• 卷：16
• 期：1
• 页码：17 - 25
• 全文大小：218K
• 年卷期：v.16,no.1(February 2000)
• ISSN：1520-6033

文摘

Numerous biopharmaceuticals and other recombinant biotechnology products are madein prokaryotic hosts. However, bacterial production of native, biologically activeeukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins.We previously described the production of soluble, native disulfide-bonded dimericproteins 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 producedwith our expression system (Garces et al., 1997). However, plasmid instability duringinduction limited its usefulness. We now report the stable, high-level expression ofsoluble, disulfide-bonded human uteroglobin without antibiotic selection. We designeda new vector containing a multifunctional stabilization region that confers completeplasmid stability and increased protein yields without copy number increases.Recombinant expression remains fully inducible after long-term continuous culturein nonselective liquid medium (at least 260 generations). This system may significantlyexpand 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.