Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli.
In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.
Sletta
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SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, Norway.
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Aune
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Hellebust
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Hakvåg
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Aune
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Ellingsen
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Valla
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Brautaset
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D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006241:Haptens; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D010081:Oxazolone / Q000031:analogs & derivatives*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic
D003470:Culture Media; D006241:Haptens; D007128:Immunoglobulin Fragments; C018361:2-phenyloxazolone; D010081:Oxazolone