title: Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.
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.
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Siti Aisyah
authors.initials: SA
authors.lastname: Mualif
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Sin-Yeang
authors.initials: SY
authors.lastname: Teow
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Tasyriq Che
authors.initials: TC
authors.lastname: Omar
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Yik Wei
authors.initials: YW
authors.lastname: Chew
authors.affiliation: Regenerative Medicine, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Narazah Mohd
authors.initials: NM
authors.lastname: Yusoff
authors.affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Ali
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D012343:RNA, Transfer