Expression of the rat interferon-α1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region

Spanjaard, Remco A.; Dijk, M. C. M. Van; Turion, A. J.; Duin, Jan van

doi:10.1016/0378-1119(89)90298-9

Cited by 24 publications

(14 citation statements)

References 20 publications

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“…High level expression of Rev was dependent on the translational coupling of the Rev to the &glucuronidase gene in a two cistron construction. The translational coupling approach has been used to overexpress several heterologous proteins in E. coli (Spanjaard et al, 1989;Schoner et al, 1984;Makoff et al, 1990) and provides a convenient method to overcome poor translational efficiency of a cloned gene. HIV-1 recombinant Rev has been expressed in high quantity as a soluble protein in E. coli and purified to greater than 95% purity.…”

Section: G T G-g a G-g A T-g A T-t A A-a T G-g Cmentioning

confidence: 99%

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

Daly

Doten²,

Rennert³

et al. 1993

Biochemistry

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Recombinant HIV-1 Rev protein was overexpressed in Escherichia coli using translational coupling to the beta-glucuronidase gene and demonstrated to interact with high affinity and specificity with the Rev responsive element (RRE). A complex Rev-dependent binding pattern was observed using the gel shift assay which could be simplified to one or two primary bands in the presence of stoichiometric concentrations of RRE. Competition of these bands with a series of homopolymer RNA species demonstrated that Rev is essentially a poly-G binding protein, although poly-I was also shown to compete for specific RRE binding. The stoichiometry of the Rev-dependent gel shift complexes was determined using 125I-labeled Rev. The stable, lowest mobility complex was determined to possess a ratio of between 7 and 8 Rev molecules per RRE containing RNA fragment while the two fastest migrating complexes contained ratios of one and two Rev molecules per RRE, respectively. Using the Hill equation as a model for cooperative interactions, a Hill coefficient of n(app) = 2 was obtained from fitting of direct nitrocellulose filter binding assays, reflecting cooperatively bound Rev molecules on the RRE under equilibrium binding conditions. An increase in ionic strength from 0.0 to 0.3 M NaCl reduced cooperative Rev binding to the RRE, but specificity of Rev for the RRE relative to antisense RNA was increased 100,000-fold. At molar ratios of Rev to RRE above 2, Rev dissociated from the RRE with a T1/2 of approximately 20-25 min.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: G T G-g a G-g A T-g A T-t A A-a T G-g Cmentioning

confidence: 99%

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

Daly

Doten²,

Rennert³

et al. 1993

Biochemistry

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“…Although the versatility of bicistronic systems has been questioned [19] a number of bicistronic vectors have been successfully used to express foreign genes whose expression was originally poor in E. coli [14,18,19,37,38]. We show here that the bicistronic system is particularly useful to achieve expression of soluble and active proteins, which carry the proline residue at the P 1 ′ position and require a free N-terminus.…”

Section: Discussionmentioning

confidence: 87%

“…Several two-cistron expression systems have been successfully applied to enhance translation efficiency thus achieving high-level expression of mammalian and archaeal genes [14–16,18–20,37–39]. To test if secondary structure at the TIR was responsible for the poor expression of MtuNei2 in E. coli , we designed a bicistronic vector in which a leader sequence or an open reading frame (ORF) preceded the target gene and included the RBS for the downstream gene (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A series of two-cistron plasmids (bicistronic vectors) have been successfully used to overcome translational inhibition of mRNAs with stable secondary structures [14–18]. In such a system, the first/upstream cistron is generally an A/T-rich sequence, which can minimize local secondary structure(s) thereby allowing efficient translation initiation [15,16], while the second/downstream cistron containing the coding sequence of the target gene is translationally coupled to the first/upstream cistron.…”

Section: Introductionmentioning

confidence: 99%

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A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

Guo

Wallace

Bandaru

2009

Protein Expression and Purification

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A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P 1 ′ position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P 1 ′ position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.

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“…coli expression vector pIF.D [11], which is derived from pPLc236 [10]. pIF.D contains a synthetic ribosomal binding site that adds a methionine to the N-terminus of mature IFN-oel.…”

Section: Construction Of Mutants Of Rat Ifn-almentioning

confidence: 99%

The cysteines in position 1 and 86 of rat interferon‐α₁ are indispensable for antiviral activity

1989

Self Cite

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The human, bovine, murine and rat interferon (IFN)-~t families contain 4 conserved cysteines located at positions 1, 29, 99 and 139 that are involved in disulfide bridges. Rat and murine IFN-~t subspecies carry a fifth Cys (Cys-86) which is not conserved in bovine and human IFN-ct subspecies except for human IFN-cfi. Changing Cys-86 in rat IFN-cq into Ser or Tyr virtually abolished antiviral activity. As shown by others, the substitution of Cys-86 to Ser in human IFN-cq had no pronounced effect on activity. This suggests that in contrast to human and bovine IFN-~t, Cys-86 in rodent IFN-ct plays a crucial role in receptor binding. Changing Cys-I to Gly in rat IFN-cq also destroyed activity, in agreement with results obtained in the human IFN-cq system.

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Expression of the rat interferon-α1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region

Cited by 24 publications

References 20 publications

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

The cysteines in position 1 and 86 of rat interferon‐α₁ are indispensable for antiviral activity

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Expression of the rat interferon-α1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region

Cited by 24 publications

References 20 publications

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element

A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

The cysteines in position 1 and 86 of rat interferon‐α1 are indispensable for antiviral activity

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The cysteines in position 1 and 86 of rat interferon‐α₁ are indispensable for antiviral activity