Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

Wolfrum, Alexandra; Brock, Stephan; Mac, Thi Ha; Grillenbeck, Norbert

doi:10.1016/s1046-5928(03)00003-2

Cited by 8 publications

(10 citation statements)

References 34 publications

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“…ArfA, RF2 and tRNA fMet were cloned from E. coli K12 strain 30 and were purified 4 . E. coli IF1, IF2, and IF3 were overexpressed and purified 31 . The mRNA with the sequence 5′-GGC AAG GAG GUA AAA AUG -3′ (P-site is underlined) was purchased from Dharmacon (Amersham/GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome

Zeng

Chen

Remis

et al. 2017

Nature

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Quality-control mechanisms intervene appropriately when defective translation events occur to preserve the intergrity of protein synthesis. Rescuing the ribosome translating on mRNAs absent of a stop codon is one of the co-translational quality control pathways in the cell. In many bacteria, ArfA recognizes stalled ribosome and recruits release factor RF2, which catalyzes the termination of protein synthesis 1–3. While an induced-fit mechanism of ArfA/RF2-mediated nonstop mRNA surveillance has been reported 4, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report a cryoEM structure of ArfA and RF2 in complex with the 70S ribosome that is bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to promote RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Further, binding of the ArfA and RF2 induces conformational changes in the ribosomal decoding center in a way that is similar to other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 attain a catalytically competent conformation for peptide release. Insights gained from this investigation provide a framework to understand recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.

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Section: Methodsmentioning

confidence: 99%

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome

Zeng

Chen

Remis

et al. 2017

Nature

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“…E. coli IF1, IF2, and IF3 were overexpressed in BL21 (DE3) cells and purified with Ni-NTA column (Wolfrum et al 2003). N-terminal His-tags in these proteins were removed by HRV-3C protease during purifications.…”

Section: Ribosome Preparation Translation Initiation and Release Famentioning

confidence: 99%

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Zeng

Jin

2015

RNA

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Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2 m ), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k cat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the k cat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

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“…Heat release was widely used for some thermostable recombinant proteins in the initial purification steps, which contributed to the precipitation of host proteins and the reduction of the potential degradation of the target product by thermal deactivation of E. coli and its proteases [31][32][33][34] . In this study, it was revealed for the first time that heat treatment was applicable for the initial purification of TAT m -survivin (T34A), leading to a 50 % increase in the content of TAT m -survivin (T34A).…”

Section: Table 2: Effect Of Zinc Ion and Temperature On The Solubility And Overall Yield Of Tat M -Survivin (T34a)mentioning

confidence: 99%

Preparation and Evaluation of activity of Highly-soluble Anticancer Protein Drug TATm-survivin (T34A)

Zhang¹

2019

pharmaceutical-sciences

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Zhang et al.: Preparation of Soluble Protein Drug T34ATAT m -survivin (T34A), an anticancer protein drug, displayed pro-apoptotic bioactivity against various cancer cells. It was expressed in Escherichia coli as inclusion bodies. To test the bioactivity of soluble TAT msurvivin (T34A), a feasible strategy was first developed for the soluble expression and purification. Effect of zinc ion and induction temperature was investigated to improve the solubility and overall production level of TAT m -survivin (T34A). High solubility (92 %) was achieved by addition of zinc ion and temperature downshift after induction. An efficient protocol of purification was established by heat release, ammonium sulphate precipitation, anion exchange and heparin affinity chromatography. Purified TAT m -survivin (T34A) was obtained with a purity of 96 %, which inhibited the proliferation of human pancreas carcinoma cell lines SW1990. In comparison to denatured TAT m -survivin (T34A), soluble TAT m -survivin (T34A) did not exhibit higher bioactivity as expected. This study represented a novel strategy to obtain highly soluble form of TAT m -survivin (T34A) and would provide useful information for production of other soluble tat-mediated fusion proteins.

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Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

Cited by 8 publications

References 34 publications

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Preparation and Evaluation of activity of Highly-soluble Anticancer Protein Drug TATm-survivin (T34A)

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