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.
Highlights
The market for recombinant biopharmaceuticals such as antibody fragments, growth factors, hormones, and other biologically based medicines is estimated to be over $140 billion p.a., with antibody products accounting for a large proportion of these sales (Walsh, 2014)
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
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)
Summary
The market for recombinant biopharmaceuticals such as antibody fragments, growth factors, hormones, and other biologically based medicines is estimated to be over $140 billion p.a., with antibody products accounting for a large proportion of these sales (Walsh, 2014). The Tat pathway has an inbuilt “quality control” system, whereby it preferentially exports correctly folded proteins—it has been shown to secrete a range of heterologous proteins, including several biopharmaceuticals (Alanen et al, 2015; Matos et al, 2014), yet quantitatively reject virtually every misfolded protein tested to date (DeLisa et al, 2003; Matos, Robinson, & Di Cola, 2008; Richter & Bruser, 2005; Robinson et al, 2011) It has potential for the export of correctly folded, highly active target proteins with minimal heterogeneity; that is, it simultaneously provides both a means of exporting the product to the periplasm and increasing product “quality,” thereby decreasing DSP costs.
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