Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins

Sommer, Benjamin; Friehs, Karl; Flaschel, Erwin

doi:10.1016/j.jbiotec.2009.11.019

Cited by 26 publications

(19 citation statements)

References 38 publications

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“…The cells were resuspended and separated into different fractions based on the method of Sommer et al (2010). The final cell debris after sonication was dissolved in 8 mol/L urea as the insoluble cytoplasmic fraction.…”

Section: Cellular Localization Of the Expressed Thioredoxin-pedamentioning

confidence: 99%

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Liu

Han

Zhou

2011

J. Zhejiang Univ. Sci. B

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Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.

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Section: Cellular Localization Of the Expressed Thioredoxin-pedamentioning

confidence: 99%

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Liu

Han

Zhou

2011

J. Zhejiang Univ. Sci. B

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“…Therefore, applied research has increasingly focused on devising strategies to enhance the efficiency of heterologous protein secretion. Recently, Sommer et al (2009Sommer et al ( , 2010 demonstrated an effective extracellular secretion system using bacteriocin release protein (BRP) with the benefit of affinity purification using maltose binding protein (MBP). Target protein fused to MBP was translocated to the periplasmic space via the N-terminal signal-dependent Sec or Tat pathway and was then released to the medium by permeabilisation of outer membrane using BRP.…”

Section: Introductionmentioning

confidence: 99%

Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Low

Mahadi

Rahim

et al. 2010

Journal of Biotechnology

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The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

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“…Comparisons of specific activity of inulosucrase cloned into periplasmic expression vector, transformed and expressed in E. coli 16 and E. coli BL21 demonstrated that extracellular expression is achieved only in colicin producing microorganism. Various reports suggested that BRP protein is responsible for the release of colicin such as colicin A, E1, E2, K, N, U, and Y into the extracellular medium (Cascales et al, 2007;Sommer et al, 2010;Singh et al, 2012). There are reports that extracellular secretion could be achieved in E. coli strain by coexpression of a lysis-promoting protein (Sommer et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…Various reports suggested that BRP protein is responsible for the release of colicin such as colicin A, E1, E2, K, N, U, and Y into the extracellular medium (Cascales et al, 2007;Sommer et al, 2010;Singh et al, 2012). There are reports that extracellular secretion could be achieved in E. coli strain by coexpression of a lysis-promoting protein (Sommer et al, 2010). E. coli cells having the outer membrane generally do not help to secrete periplasmic proteins into the culture medium.…”

Section: Discussionmentioning

confidence: 99%

“…In some case, recombinant proteins directed to the periplasm were found in the medium but the process is not known (Choi and Lee, 2004;Mergulhao et al, 2005). Sommer et al (2010) constructed a plasmid which contains bacteriocin release proteins (BRP) that allow secretion of recombinant protein from the periplasm into the culture medium (Sommer et al, 2010). It was known that BRP or lysis proteins are responsible for the release of colicins such as A, E1, E2, K, N, U, and Y (Cascales et al, 2007;Singh et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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In vitro comparism of the extracellular secretion of inulosucrase enzyme in potential probiotic Escherichia coli 16 and BL-21

Kumar¹,

Gopalakrishnan²,

Kumar³

2013

Afr. J. Biotechnol.

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Escherichia coli 16 has potential probiotic properties including antimicrobial activity due to extracellular secretion of colicins E1/1a1b. Inulosucrase (InuJ) enzyme catalyses the polymerization of a fructose moiety of sucrose leading to the formation of fructooligosaccharides. The present investigation compared the activity of InuJ enzymes cloned into pMAL-p2ΔlacI Q a deletion vector and transformed into E. coli 16 and standard strain that is, E. coli BL21. Specific activities of InuJ enzyme were estimated in supernatant, periplasm and lysate. Specific activities of InuJ activity in cell lysate were similar in E. coli 16 and E. coli BL21 without induction of tac promoter with isopropyl thio-β-Dgalactoside (IPTG). InuJ activity is mainly present in the periplasm of E. coli BL21 whereas in E. coli 16, most of the activity is in the supernatant. Superantant of E. coli 16 strain also showed good antibacterial activity due to colicin E1/Ia1b. Colicin E1/1a1b transport system could allow extracellular secretion of InuJ proteins in probiotic E. coli 16.Key words: Colicin, extracellular, E. coli, fructooligosaccharide, inulosucrase, prebiotic, probiotic. INTRODUCTIONPrebiotics are a category of nutraceutical product that has the ability to promote the growth of specific beneficial gut bacteria (Kelly, 2008). In 2007, Roberfroid defined prebiotics as "a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gut microflora that confers health benefit". Fructooligosaccharides (FOS) has been used as prebiotic and is considered as a functional food ingredient (Cherbut, 2002;Fanaro et al., 2005;Bouhnik et al., 2006;Roberfroid, 2007;Paineau et al., 2008). FOS is the common name for fructose oligomers that are mainly composed of 1-kestose (GFS2), 2-nystose (GF3) among others in which fructose units are bound at the β-2, 1 position of sucrose through the transfructosylating enzymes such as glucosyltransferases, fructosyltransferases and inulosucrase (Yun et al., 1996). Inulosucrase has been previously shown to be involved in the synthesis of FOS (Van Hijum et al., 2006).Escherichia coli, a Gram negative bacterium is widely used as a host strain for recombinant protein production in industry. Some natural E. coli strains secrete protein extracellulally but their mechanisms of secretion are not clearly understood, nor are they widely exploited for recombinant protein production and metabolic engineering (Ni and Chen, 2009). In some case, recombinant proteins directed to the periplasm were found in the medium but the process is not known (Choi and Lee, 2004;Mergulhao et al., 2005). Sommer et al. (2010) constructed a plasmid which contains bacteriocin release proteins (BRP) that allow secretion of recombinant protein from the periplasm into the culture medium (Sommer et al., 2010). It was known that BRP or lysis proteins are responsible for the release of colicins such as A, E1, E2, K, N, U, and Y (Cascales et al., 2007;Singh et al., 2012). Previously, we had reported E. coli ...

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Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins

Cited by 26 publications

References 38 publications

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

In vitro comparism of the extracellular secretion of inulosucrase enzyme in potential probiotic Escherichia coli 16 and BL-21

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