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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

Production and purification of HIV-1 Nef and P24.NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.
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pone.0130446.g010: Production and purification of HIV-1 Nef and P24.NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.

Mentions: Bacterial cultures were grown using optimized conditions i.e. super broth (SB) as culture medium, 0.05mM IPTG as inducer, and cultivation at 22°C for 12h. Cells were harvested and optical densities at 600nm and total biomass yield were determined. Lysed bacterial pellets were used to purify Nef and P24/CA as described in Materials and Methods. Growth curves were similar between the cultures expressing Nef or P24/CA from pSA-HNef/P24-6His-RIL and pSA-HNef/P24-6His/pACYC-RIL. Cultures expressing Nef or P24 from pSA-HNef/P24-6His exhibited a lag in growth (Fig 10A and 10C) suggestive of Nef and P24 toxicity towards bacteria when overexpressed in the absence of rare tRNAs. Highly purified Nef and P24/CA were obtained from cultures expressing nef and p24 in presence of rare tRNAs (Fig 10B and 10D). Using the optimized conditions, we obtained ~5–5.5 mg highly pure (up to 84%) recombinant HV-1 Nef and P24/CA protein from each 1g biomass of pSA-HNef/P24-6His-RIL or pSA-HNef/P24-6His + pACYC-RIL –transformed NiCo21(DE3) bacteria when propagated in SB, and induced with 0.05mM IPTG for 12h at 22°C. There appeared no difference in the final yields and purity of Nef or P24 produced by single (pSA-HNef/P24-6His-RIL) and traditional double-vector (pSA-HNef/P24-6His + pACYC-RIL) system.


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)

Production and purification of HIV-1 Nef and P24.NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g010: Production and purification of HIV-1 Nef and P24.NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.
Mentions: Bacterial cultures were grown using optimized conditions i.e. super broth (SB) as culture medium, 0.05mM IPTG as inducer, and cultivation at 22°C for 12h. Cells were harvested and optical densities at 600nm and total biomass yield were determined. Lysed bacterial pellets were used to purify Nef and P24/CA as described in Materials and Methods. Growth curves were similar between the cultures expressing Nef or P24/CA from pSA-HNef/P24-6His-RIL and pSA-HNef/P24-6His/pACYC-RIL. Cultures expressing Nef or P24 from pSA-HNef/P24-6His exhibited a lag in growth (Fig 10A and 10C) suggestive of Nef and P24 toxicity towards bacteria when overexpressed in the absence of rare tRNAs. Highly purified Nef and P24/CA were obtained from cultures expressing nef and p24 in presence of rare tRNAs (Fig 10B and 10D). Using the optimized conditions, we obtained ~5–5.5 mg highly pure (up to 84%) recombinant HV-1 Nef and P24/CA protein from each 1g biomass of pSA-HNef/P24-6His-RIL or pSA-HNef/P24-6His + pACYC-RIL –transformed NiCo21(DE3) bacteria when propagated in SB, and induced with 0.05mM IPTG for 12h at 22°C. There appeared no difference in the final yields and purity of Nef or P24 produced by single (pSA-HNef/P24-6His-RIL) and traditional double-vector (pSA-HNef/P24-6His + pACYC-RIL) system.

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