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.
Bibliographical note© 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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Funding: Biotechnology and Biological Sciences Research Council. Grant Number: BB/M018288/1
- Escherichia coli/genetics
- Gene Products, tat/genetics
- Genetic Enhancement/methods
- Growth Hormone/biosynthesis
- Metabolic Networks and Pathways/genetics
- Promoter Regions, Genetic/genetics
- Recombinant Proteins/biosynthesis
- Secretory Pathway/genetics