Dataset Information

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

ABSTRACT: Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1?hr induction. TatExpress strains accumulate in excess of 30?mg?L^-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

SUBMITTER: Browning DF

PROVIDER: S-EPMC5698719 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

Browning Douglas F DF Richards Kirsty L KL Peswani Amber R AR Roobol Jo J Busby Stephen J W SJW Robinson Colin C

Biotechnology and bioengineering 20171006 12

Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, u ...[more]

PMID: 28842980

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Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

Publications

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

Similar Datasets

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