Stability studies with different vector backbones utilizing the T7 expression system in <i>Escherichia coli</i>

Walia, Rupali; Deb, J. K.; Mukherjee, Kakali

doi:10.1002/jctb.1885

Cited by 2 publications

(3 citation statements)

References 17 publications

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“…Hence, effective strategies to avoid loss of expression have been proposed previously based on preventing basal expression [38,31,33]. Therefore, the attempt to control the basal expression in the pre-induction phase should be critical for successful production of target protein [26,34,37]. …”

Section: Resultsmentioning

confidence: 99%

“…However, only basal level of T7 RNA polymerase activity can lead to substantial expression of foreign protein even without inducer [31], and such basal expression of foreign protein, especially toxic proteins, would bring about negative effects on expression stability and consequent protein production [31-33]. Therefore, lac operator would be taken into account to tightly repress expression in the pre-induction phase for reducing the transcription level of target mRNA and also the basal expression level of foreign protein by blocking both the promoter for T7 RNA polymerase synthesis in the chromosome of E. coli and T7 promoter for target protein expression in the vector [31,34,35]. …”

Section: Introductionmentioning

confidence: 99%

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High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Nie

Wang

et al. 2013

PLoS ONE

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Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Nie

Wang

et al. 2013

PLoS ONE

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“…Various bacterial promoters like Tac, T7, Trp, lacUV5 and araBAD have been widely used (Guzman et al 1995;Cronan 2006) and the pBAD series of vectors with araBAD promoter are routinely used for heterologous protein expression in E. coli (Cronan 2006). The arabinose operon system offers advantages like protein expression using an inexpensive inducer, L-arabinose, use of common E. coli hosts like DH5a, XL-1 Blue, JM109 for protein expression and higher plasmid stability (Walia et al 2008). This promoter system has also been introduced into both Grampositive and Gram-negative bacterial hosts (Newman and Fuqua 1999;Sukhchawalit et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

et al. 2009

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A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

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Stability studies with different vector backbones utilizing the T7 expression system in Escherichia coli

Abstract: BACKGROUND: The T7 system is used ubiquitously for high-level expression of recombinant proteins in lab scale cultures of Escherichia coli. However, its functional stability during scale-up is critical for it to be useful in industrial scale fermentations.

Cited by 2 publications

References 17 publications

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

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