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Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

Mualif SA, Teow SY, Omar TC, Chew YW, Yusoff NM, Ali SA - PLoS ONE (2015)

Bottom Line: These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids.To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector.We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

View Article: PubMed Central - PubMed

Affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.

ABSTRACT
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

No MeSH data available.


Related in: MedlinePlus

Optimization of IPTG concentration and induction temperature.(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).
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pone.0130446.g003: Optimization of IPTG concentration and induction temperature.(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).

Mentions: In order to optimize Nef expression, we tested ranges of IPTG concentrations (0–0.4mM) and incubation temperatures (30, 22, and 18°C). Expression of HIV-1 Nef was optimal at the lowest tested concentration of IPTG i.e. 0.05mM as determined by the western blot analysis (Fig 3A). HIV-1 Nef production was similar in 0.05mM IPTG-induced cultures when grown at 30°C and 22°C, but lower in 18°C-grown cultures (Fig 3B). However, neither IPTG concentration nor various incubation temperatures enhanced overall Nef production in E. coli NiCo21(DE3) cells.


Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

Mualif SA, Teow SY, Omar TC, Chew YW, Yusoff NM, Ali SA - PLoS ONE (2015)

Optimization of IPTG concentration and induction temperature.(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492947&req=5

pone.0130446.g003: Optimization of IPTG concentration and induction temperature.(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).
Mentions: In order to optimize Nef expression, we tested ranges of IPTG concentrations (0–0.4mM) and incubation temperatures (30, 22, and 18°C). Expression of HIV-1 Nef was optimal at the lowest tested concentration of IPTG i.e. 0.05mM as determined by the western blot analysis (Fig 3A). HIV-1 Nef production was similar in 0.05mM IPTG-induced cultures when grown at 30°C and 22°C, but lower in 18°C-grown cultures (Fig 3B). However, neither IPTG concentration nor various incubation temperatures enhanced overall Nef production in E. coli NiCo21(DE3) cells.

Bottom Line: These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids.To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector.We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

View Article: PubMed Central - PubMed

Affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.

ABSTRACT
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

No MeSH data available.


Related in: MedlinePlus