An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100

Bao, Ru-Meng; Yang, Hong-Ming; Yu, Chang-Mei; Zhang, Weifen; Tang, Jin‐Bao

doi:10.1016/j.pep.2016.05.007

Cited by 29 publications

(22 citation statements)

References 40 publications

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“…S2). While glycine induces swelling E. coli cells and enlargement of the periplasmic space by interfering with peptidoglycan synthesis, Triton X-100 disrupts the integrity of the outer membrane (Yang et al 1998;Bao et al 2016). Triton X-100 did not significantly improve the refolding yield of scFv-BM3 ( Fig.…”

Section: Extracellular Secretion Of Functional Scfv-bm3mentioning

confidence: 95%

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Kim²,

Choi³

et al. 2019

Lett Appl Microbiol

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scFv‐BM3 is a single‐chain variable fragment (scFv) against aflatoxin B1 (AFB1) engineered by affinity maturation and site‐directed mutagenesis, and thus has a 31‐fold higher affinity than its wild‐type. To apply scFv‐BM3 to immunological detection of AFB1, periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv‐BM3. scFv‐BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv‐BM3 secreted into the culture medium had binding activity to AFB1. Expression conditions for scFv‐BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv‐BM3 was significantly improved by supplementation with Triton X‐100 and optimization of expression conditions. The scFv‐BM3 purified from the culture medium exhibited a typical antiparallel β‐sheet structure and adopted a proper conformation to bind AFB1 with high affinity and specificity in various biophysical and biochemical analyses. Significance and Impact of the Study Single‐chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B1 (AFB1) (scFv‐BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv‐BM3 resulted in formation of inactive aggregates in E. coli, the scFv‐BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB1. This study promotes the application of functional scFv‐BM3 to the immunological detection of AFB1 in biotechnology fields.

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Section: Extracellular Secretion Of Functional Scfv-bm3mentioning

confidence: 95%

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Kim²,

Choi³

et al. 2019

Lett Appl Microbiol

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“…Both were used in various attempts to increase secretion, however, both exhibit negative effects on cell growth and/or increased cell lysis. Extracellular production of a recombinant fusion protein between ZZ protein and alkaline phosphatase could be improved by a factor of 18 without apparent lysis by synergistic effects of 5% sucrose, 1% glycine, and 1% Triton X-100 [231]. In another case, supplementation of the medium with 0.5% Triton X-100 resulted in a 48-fold increased secretion of a pullulanase from Bacillus deramificans, compared to the control without supplementation [229].…”

Section: Cultivation/fermentationmentioning

confidence: 97%

“…Both supplements were also combined (2% glycine + 1% Triton X-100) to increase secretion, leading to a 170-fold increased excretion of an extracellular fusion protein with a single chain antibody and tumor necrosis factor (sFV/TNF-alpha) [230]. Extracellular production of a recombinant fusion protein between ZZ protein and alkaline phosphatase could be improved by a factor of 18 without apparent lysis by synergistic effects of 5% sucrose, 1% glycine, and 1% Triton X-100 [231].…”

Section: Cultivation/fermentationmentioning

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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“…A potential solution is to increase butyrate uptake by the addition of cell permeabilization agents (e.g. Triton X-100 and / or sucrose) 16 but this led to only a modest increase in propane titres (1.6-fold; Table S4). Another approach is to stimulate the butyrate uptake transporter atoE (part of the atoDAEB small chain fatty acid catabolism operon) with acetoacetate.…”

Section: <Insert Figure 3 Here>mentioning

confidence: 99%

Distributed Biomanufacturing of Liquefied Petroleum Gas

Hoeven

Jmx

Amer

et al. 2019

Preprint

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Liquefied Petroleum Gas (LPG) is a major domestic and transport fuel. Its combustion lessens NO x , greenhouse gas and particulates emissions compared to other fuels. Propane -the major constituent of LPG -is a clean, high value 'drop-in' fuel that can help governments develop integrated fuels and energy policies with low carbon burden, providing solutions to the multi-faceted challenges of future energy supply. We show that bio-LPG (bio-propane and bio-butane) can be produced by microbial conversion of waste volatile fatty acids that can be derived from anaerobic digestion, industrial waste, or CO 2 via photosynthesis. Bio-LPG production was achieved photo-catalytically, using biomass propagated from bioengineered bacteria including E. coli, Halomonas (in non-sterile seawater), and Synechocystis (photosynthetic). These fuel generation routes could be implemented rapidly in advanced and developing nations of the world to meet energy needs, global carbon reduction targets and clean air directives.[141words]

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An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100

Cited by 29 publications

References 40 publications

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Secretion of recombinant proteins from E. coli

Distributed Biomanufacturing of Liquefied Petroleum Gas

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An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100

Cited by 29 publications

References 40 publications

Extracellular production of functional single-chain variable fragment against aflatoxin B1usingEscherichia coli

Extracellular production of functional single-chain variable fragment against aflatoxin B1usingEscherichia coli

Secretion of recombinant proteins from E. coli

Distributed Biomanufacturing of Liquefied Petroleum Gas

Contact Info

Product

Resources

About

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli