Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter

Beshay, Usama; Miksch, Gerhard; Friehs, Karl; Flaschel, Erwin

doi:10.1007/s10529-007-9477-4

Cited by 13 publications

(12 citation statements)

References 18 publications

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“…Since the expression of kil could affect the viability of the cells, the induction time and the promoter strength for Kil expression are crucial. Using Bacillus b-glucanase as a model protein, Beshay et al (2007) investigated four expression vectors harboring all possible combinations of a weak and a strong stationary-phase promoter for the kil and b-glucanase genes, respectively. Despite of the potential negative influence of kil gene on the cell growth, higher extracellular b-glucanase was reached when the expression of b-glucanase was regulated by a strong promoter, in which over 90% of the total b-glucanase was secreted into the medium.…”

Section: Using Cell Envelope Mutantsmentioning

confidence: 99%

Extracellular recombinant protein production from Escherichia coli

Chen

2009

Biotechnol Lett

124

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Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

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Section: Using Cell Envelope Mutantsmentioning

confidence: 99%

Extracellular recombinant protein production from Escherichia coli

Chen

2009

Biotechnol Lett

124

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“…Since four strains with different promoter combinations for b-glucanase production and secretion (Beshay et al 2007b) were considered, 16 media-strain combinations were tested (see Figs. 1, 2).…”

Section: Resultsmentioning

confidence: 99%

“…Four plasmids, the construction of which was published in detail elsewhere (Beshay et al 2007b), were transformed into Escherichia coli JM109. Each carried the kil gene and a gene for a hybrid b-glucanase H1 (Borriss et al 1989) but under control of different promoters.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Screening for conditions of enhanced production of a recombinant β-glucanase secreted into the medium by Escherichia coli

et al. 2009

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The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (>200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

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“…Colicin E1 BRP was already used for extracellular production of alkaline phosphatase [200,202], phytase [203], penicillin amidase [77], and β-glucanase [204,205]. The yields of extracellular proteins depend on the promoter strength for BRP and target protein expression, induction time [200,[202][203][204], dependency of Sec-or Tat-pathway for translocation into the periplasm [115], and medium composition [205]. Extracellular proteins that have been produced by the coexpression of this BRP are alkaline phosphatase [200,202], β-lactamase [200], maltose-binding protein, hybrids thereof, and also Tat-dependent GFP [115].…”

Section: Induced Permeabilitymentioning

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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Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter

Cited by 13 publications

References 18 publications

Extracellular recombinant protein production from Escherichia coli

Extracellular recombinant protein production from Escherichia coli

Screening for conditions of enhanced production of a recombinant β-glucanase secreted into the medium by Escherichia coli

Secretion of recombinant proteins from E. coli

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