<i>Escherichia coli</i> “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Browning, Douglas F.; Richards, Kirsty L; Peswani, Amber R; Roobol, Jo; Busby, Stephen J. W.; Robinson, Colin

doi:10.1002/bit.26434

Cited by 46 publications

(53 citation statements)

References 24 publications

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“…In this pathway, the protein cargo must be either completely or partially unfolded to be secreted. In the case of secretion of peptides or smaller proteins, studies have reported extremely high production (Browning et al, 2017;Chen et al, 2014), for example, 6 g/L of 13.1-kDa Fab (Voulgaris, Finka, Uden, & Hoare, 2015). Small proteins or peptides are secreted into the periplasm by the Type II secretion system with extracellular secretion using a leaky outer membrane.…”

Section: Possibility Of Omvs As a Tool For Protein Secretionmentioning

confidence: 99%

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Ojima

Sawabe

Konami

et al. 2019

Biotech & Bioengineering

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Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.

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Section: Possibility Of Omvs As a Tool For Protein Secretionmentioning

confidence: 99%

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Ojima

Sawabe

Konami

et al. 2019

Biotech & Bioengineering

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“…The twin-arginine translocase offers the potential for the biotechnology industry owing to its ability to transport fully-folded proteins into the periplasm of E. coli. Initial studies confirmed the capability of the Tat system to transport a model heterologous protein to a high yield (Matos et al, 2012), and more recent data has shown that substrates are not limited to GFP with Tat being able to transport hGH, scFv, and interferon α2b (Alanen et al, 2015;Browning et al, 2017).…”

Section: Discussionmentioning

confidence: 85%

“…Initial studies confirmed the capability of the Tat system to transport a model heterologous protein to a high yield (Matos et al, 2012), and more recent data has shown that substrates are not limited to GFP with Tat being able to transport hGH, scFv, and interferon α2b (Alanen et al, 2015;Browning et al, 2017). Initial studies confirmed the capability of the Tat system to transport a model heterologous protein to a high yield (Matos et al, 2012), and more recent data has shown that substrates are not limited to GFP with Tat being able to transport hGH, scFv, and interferon α2b (Alanen et al, 2015;Browning et al, 2017).…”

Section: Discussionmentioning

confidence: 85%

“…Most of the Tat system's substrates are soluble proteins, and most studies on the biotechnological exploitation of the system have similarly focused on soluble high-value protein (Alanen et al, 2015;Browning et al, 2017;Matos et al, 2013). However, the Tat system does have considerable potential for the production of single-span heterologous membrane proteins, particularly those that contain a globular domain located on the periplasmic face of the inner membrane.…”

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confidence: 99%

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Characterization of a novel method for the production of single‐span membrane proteins in Escherichia coli

Smith

Walker

Jones

et al. 2019

Biotech & Bioengineering

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The large‐scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high‐value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin‐arginine translocase, to transport fully‐folded proteins into the periplasm of not only E. coli, but also other Gram‐negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single‐span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno‐electron microscopy approaches were used to investigate the export of human growth hormone by the twin‐arginine translocase, and the generation of a single‐span membrane‐embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single‐span membrane proteins in E. coli.

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“…No negative impact on cell growth or target integrity was detected [94], but the translocation is limited by the PMF, which is the main energy source of the translocon. [95] An additional coexpression of phage-shock protein A (PspA), which maintains the PMF, can further increase the translocation efficiency [96]. Placing ptac upstream of the chromosomal tatABCD operon has also been shown to facilitate Tat export.…”

Section: Tat-pathwaymentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Cited by 46 publications

References 24 publications

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Characterization of a novel method for the production of single‐span membrane proteins in Escherichia coli

Secretion of recombinant proteins from E. coli

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