Robotic Cloning and Protein Production Platform of the Northeast Structural Genomics Consortium

Acton, Thomas; Gunsalus, Kristin C.; Xiao, Rong; Chung, Li; Aramini, James M.; Baran, M.C.; Chiang, Yiwen; Climent, Teresa; Cooper, B.; Denissova, Natalia G.; Douglas, Shawn M.; Everett, J.K.; Ho, C.K.; Macapagal, Daphne; Rajan, P.K.; Shastry, R.; Shih, Liang Yu; Swapna, G.V.T.; Wilson, Michael; Wu, M.J.; Gerstein, Mark; Inouye, Masayori; Hunt, J.F.; Montelione, Gaetano T.

doi:10.1016/s0076-6879(05)94008-1

Cited by 123 publications

(137 citation statements)

References 55 publications

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“…(A) GST-pulldown assay. GST-F2F3 or GST were mixed with equal amounts of the WT or indicated mutant 35 S-labeled full-length NS1A protein of Ud, which were prepared as described in SI Materials and Methods. The labeled proteins eluted with glutathione from GST-F2F3 or GST were resolved by SDSpolyacrylamide gels, which were analyzed by exposure to X-ray film.…”

Section: Resultsmentioning

confidence: 99%

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Structural basis for suppression of a host antiviral response by influenza A virus

Das

Chung

Xiao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-␤ mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3 end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-␤ pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.antiviral drug discovery ͉ bird flu ͉ vaccine engineering ͉ virology ͉ X-ray crystallography

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Section: Resultsmentioning

confidence: 99%

“…NS1A effector domain NS1A (85-215) and the F2F3 (60-120) fragments of CPSF30, were cloned into modified pET21c and pET14c (Novagen) vectors (35). The constructs were verified by DNA sequence analysis and expressed in E. coli BL21(DE3) cells containing the rare tRNA expression plasmid pMGK.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for suppression of a host antiviral response by influenza A virus

Das

Chung

Xiao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

201

288

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“…44,45 The full-length protein was cloned into a pET21b vector, along with a S28F mutation and a short noncleavable C-terminal hexa His tag. The transformed cells were cultured at 37 C in MJ9 minimal medium 45 containing ( 15 NH 4 ) 2 SO 4 and U-13 C-glucose as the sole nitrogen and carbon sources, respectively.…”

Section: Expression and Purification Of Ser13mentioning

confidence: 99%

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

et al. 2010

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The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra-and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra-or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K d 3.4 6 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.

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“…One of the major consequences of the structural genomics revolution has been the implementation of high-throughput construct screening. [2][3][4][5][6] In these genome-wide efforts large number of proteins undergo initial screening on a small scale (0.1-0.5 mL) to determine expression level and solubility. 4,5 The folding state and solution behavior of high yield targets are then often evaluated by solution NMR spectroscopy through the acquisition of [ 1 H, 15 N]-correlation spectra.…”

Section: Introductionmentioning

confidence: 99%

“…4,5 The folding state and solution behavior of high yield targets are then often evaluated by solution NMR spectroscopy through the acquisition of [ 1 H, 15 N]-correlation spectra. [2][3][4][5][6][7] This type of spectrum provides a ''fingerprint'' of the protein which informs on the suitability of a sample for structural studies and also on the degree of folding of the protein. [8][9][10] Taking advantage of these improvements, we have developed a protocol that allows rapid evaluation of the feasibility of structural studies for multiple protein targets expressed in parallel in E. coli.…”

Section: Introductionmentioning

confidence: 99%

Parallel screening and optimization of protein constructs for structural studies

et al. 2009

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A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X-ray crystallographic investigations.

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Robotic Cloning and Protein Production Platform of the Northeast Structural Genomics Consortium

Cited by 123 publications

References 55 publications

Structural basis for suppression of a host antiviral response by influenza A virus

Structural basis for suppression of a host antiviral response by influenza A virus

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

Parallel screening and optimization of protein constructs for structural studies

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