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

Banerjee, Sampali; Salunkhe, Shardul S.; Apte-Deshpande, Anjali; Mandi, Naganath; Mandal, Gautam; Padmanabhan, Sriram

doi:10.1007/s10529-009-9976-6

Cited by 12 publications

(9 citation statements)

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“…An E. coli expression system, which is the most common type of expression system, was used in the present study (Banerjee et al 2009;Small et al 2010;Sorensen and Mortensen 2005). The E. coli expression system is preferred because of its low cost, high productivity and easy operation.…”

Section: Introductionmentioning

confidence: 99%

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

Xiao

Hao

Wang

et al. 2014

Geomicrobiology Journal

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Molecular mechanisms and gene regulation are of interest in the area of geomicrobiology in which the interaction between microbes and minerals is studied. This paper focuses on the regulation of the expression of carbonic anhydrase (CA) genes in Bacillus mucilaginosus and the effects of the expression product of the B. mucilaginosus CA gene in Escherichia coli on calcite weathering. Real-time fluorescent quantitative PCR (RT-qPCR) was used to explore the relationship between CA gene expression in B. mucilaginosus and promotion of calcite dissolution under condition of Ca 2C deficiency. The results showed that adding calcite to the medium, which lacks Ca 2C , can up-regulate the expression of the bacterial CA genes to accelerate calcite dissolution for bacterial growth. CA genes from B. mucilaginosus were transferred into E. coli by cloning. We then employed crude enzyme extract from the resultant E. coli strain in calcite dissolution experiments. The enzyme extract promoted calcite dissolution. These findings provide direct evidence for the role of microbial CA on mineral weathering and mineral nutrition release.

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Section: Introductionmentioning

confidence: 99%

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

Xiao

Hao

Wang

et al. 2014

Geomicrobiology Journal

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“…Since pBAD24M vector could be easily used for hyper expression of heterologous proteins in E. coli K12 host due to the presence of enhancer elements as described before [38], we decided to clone the gene coding for GFPuv into this vector and examine if the difference in the GFP fluorescence was due to difference in the amount of GFP expression per cell rather than the stability of the protein. The GFPuv was PCR amplified from pGFPuv vector using GFP specific primers and cloned into EcoR1/HindIII sites as described earlier by Banerjee et al [38].…”

Section: Methodsmentioning

confidence: 99%

“…The GFPuv was PCR amplified from pGFPuv vector using GFP specific primers and cloned into EcoR1/HindIII sites as described earlier by Banerjee et al [38]. This plasmid was introduced into competent cells of E. coli K12 and E. coli B strains and GFP expressions were carried out in plain Luria Bertani broth (LB).…”

Section: Methodsmentioning

confidence: 99%

“…The primers used were forward primer as 5'-CCG CCG GAA TTC GAT ATC ATG AGT AAA GGA GAA GAA CTT TTC -3' and reverse primer as 5'-CCG CCG GAA TTC TTA TTT GTA GAG CTC ATC CAT GCC -3'. The PCR product was cloned into pBAD24M vector [38] at the EcoRI site. The correct orientation of clones was confirmed by EcoRV digestion and tested for expression in suitable E. coli host.…”

Section: Methodsmentioning

confidence: 99%

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Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

et al. 2010

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BackgroundThe green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.ResultsWe describe the construction of two GFP variants with modified putative OmpT proteolytic sites by site directed mutagenesis (SDM). Such modified genes upon arabinose induction exhibited varied degrees of GFP fluorescence. While the mutation of K79G/R80A (SDM I) resulted in dramatic loss of fluorescence activity, the modification of K214A/R215A (SDM II) resulted in four fold enhanced fluorescence of GFP.ConclusionsThis is the first report on effect of OmpT protease site modification on GFP fluorescence. The wild type and the GFP variants showed similar growth profile in bioreactor studies with similar amounts of recombinant GFP expressed in the soluble fraction of the cell. Our observations on higher levels of fluorescence of SDM II GFP mutant over native GFPuv in an OmpT+ host like DH5α, JM109 and LE392 at 37°C reiterates the role played by host OmpT in determining differences in fluorescent property of the expressed GFP. Both the WT GFP and the SDM II GFP plasmids in E. coli BL21 cells showed similar expression levels and similar GFP fluorescent activity at 37°C. This result substantiates our hypothesis that OmpT protease could be a possible factor responsible for reducing the expression of GFP at 37°C for WT GFP clone in K12 hosts like DH5α, JM109, LE 392 since the levels of GFP expression of SDM II clone in such cells at 37°C is higher than that seen with WT GFP clone at the same temperature.

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“…The recombinant production of enzymes in easily cultivable hosts is a suitable means to achieve high productivity. Escherichia coli is a commonly used host for heterologous protein expression because of its fast growth and easy genetic manipulation (Banerjee et al 2009). To date, several bacterial laccases have been successfully expressed in E. coli (Santhanam et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater

Zhang

Song

et al. 2015

Environ Sci Pollut Res

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Fungal laccases are typically unstable at high pH and temperature conditions, which limit their application in the decolorization of textile wastewater. By contrast, the highly stable bacterial laccases can function within a wider pH range and at high temperatures, thus have significant potential in treatment for textile wastewater. In our previous work, a thermo-alkali-stable CotA-laccase gene was cloned from Bacillus pumilus W3 and overexpressed in Escherichia coli. In this study, the robust CotA-laccase achieved efficient secretory expression in Bacillus subtilis WB600 by screening a suitable signal peptide. A maximum CotA-laccase yield of 373.1 U/mL was obtained at optimum culture conditions in a 3-L fermentor. Furthermore, the decolorization and detoxification of textile industry effluent by the purified recombinant CotA-laccase in the presence and absence of redox mediators were investigated. Among the potential mediators that enhanced effluent decolorization, acetosyringone (ACS) was the most effective. The toxicity of the CotA-laccase-ACS-treated effluent was greatly reduced compared with that of the crude effluent. To the best of our knowledge, this study is the first to report on the heterologous expression of CotA-laccase in B. subtilis. The recombinant strain B. subtilis WB600-5 has a great potential in the industrial production of this bacterial enzyme, and the CotA-laccase-ACS system is a promising candidate for the biological treatment of industrial textile effluents.

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

Cited by 12 publications

References 14 publications

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater

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

Cited by 12 publications

References 14 publications

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca2+Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca2+Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater

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The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution

The Up-regulation of Carbonic Anhydrase Genes ofBacillus mucilaginosusunder Soluble Ca²⁺Deficiency and the Heterologously Expressed Enzyme Promotes Calcite Dissolution