Overcoming Expression and Purification Problems of RhoGDI Using a Family of “Parallel” Expression Vectors

Sheffield, Peter; Garrard, Sarah; Derewenda, Zygmunt S.

doi:10.1006/prep.1998.1003

Cited by 615 publications

(498 citation statements)

References 20 publications

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“…The cells were incubated at a steady temperature of 25 ºC for 24 h, collected by centrifugation at 3,382 × g for 10 min, and suspended in default buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) with the complete VncS protein (residues 1–442) or with the kinase domain of VncS (residues 194–442) with 0.5% Triton X-100. The cells weresubsequently lysed by sonication on ice, and the cellular debris and insoluble proteins were removed by centrifugation at 16,000 × g for 1 h. The protein fractions in the supernatant were purified using a Ni-NTA resin (Qiagen) as previously described [41]. When necessary, the His 6 tag was removed using 100 units of the tobacco etch virus protease during dialysis against 1 liter of default buffer along with 1 mM EDTA and 1 mM dithiothreitol overnight at 4 ºC.…”

Section: Methodsmentioning

confidence: 99%

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia

et al. 2018

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Streptococcus pneumoniae (pneumococcus), the major pathogen for pneumonia, commonly colonizes the lung, but the mechanism underlying the coordination of virulence factors during invasion via the host protein remains poorly understood. Bacterial lysis releases the components of the cell wall, and triggers innate immunity and the subsequent secretion of pro-inflammatory cytokines. Previously, the virulence of the pep27 mutant was shown to be attenuated as a feasible candidate for vaccine development. However, the role of pep27 gene, belonging to the vancomycin-resistance locus (vncRS operon), in virulence, is largely unknown. This study demonstrates that transferrin in the host serum reduces the survival of the host during S. pneumoniae infections in mice. The exposure of the pneumococcal D39 strain to lactoferrin induced the vncRS operon, lysis, and subsequent in vivo cytokine production, resulting in lung inflammation. However, these responses were significantly attenuated in pneumococci harboring a mutation in pep27. Mechanistically, the VncS ligand, identified as lactoferrin, induced the vncRS operon and increased the in vivo mortality rates. Thus, serum-induced activation of vncRS plays an essential role in inducing pneumonia.

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Section: Methodsmentioning

confidence: 99%

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia

et al. 2018

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“…The Nup37 cDNA was subcloned as an EcoRI/XbaI fragment in the pGST-parallel3 vector (Sheffield et al, 1999) to produce the recombinant fusion protein GST-Nup37, and in pCS2ϩMT vector (Turner and Weintraub, 1994) to express 6-myc tagged fusion. Plasmid pEGFP 3 -Sec13 was constructed by inserting the Sec13 ORF (provided by B. Glick, University of Chicago, Chicago, IL) in frame in the pEGFP 3 -C vector.…”

Section: Plasmidsmentioning

confidence: 99%

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

Loïodice

Alves

Rabut

et al. 2004

MBoC

254

240

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In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

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“…To improve expression, the genes encoding 6His-tagged bovine Ga-(s) (short) and Ga-(i2) were recloned from pML1213HisGs and MLHisGi2 plasmids (a gift from Dr. Maurine Linder, Cornell University, Ithaca, NY) into pHis1 plasmid (31) to yield the pHis-Gs and pHis-Gi2 plasmids, respectively. The G proteins were expressed and purified from Escherichia coli (BL21-DE3) as described previously (32) and stored in 50 mM HEPES buffer, pH 8.0, 0.5 mM EDTA, 2 mM dithiothreitol (DTT), 50 mM guanosine diphosphate (GDP), and 10% glycerol at À20 C under argon.…”

Section: Methodsmentioning

confidence: 99%

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain

Press

Zvagelsky

Vyazmensky

et al. 2016

Biophysical Journal

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The signaling of a G protein-coupled receptor (GPCR) is dictated by the complementary responsiveness of interacting intracellular effectors such as G proteins. Many GPCRs are known to couple to more than one G protein subtype and induce a multitude of signaling pathways, although the in vivo relevance of particular pathways is mostly unrecognized. Dissecting GPCR signaling in terms of the pathways that are activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. Here, we describe the design of soluble GPCR mimetics to study the details of the interplay between G-proteins and activators. We constructed functional mimetics of the intracellular domain of a model GPCR, the thyrotropin receptor. We based the construction on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six a-helices. The 6-Helix scaffold, which endowed the substituted thyrotropin receptor intracellular domain elements with spatial constraints analogous to those found in native receptors, enabled the reconstitution of a microdomain that consists of intracellular loops 2 and 3, and is capable of binding and activating Ga-(s). The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could help investigators develop novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and counteracting cellular dysfunctions via focused tuning of GPCR signaling.

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Overcoming Expression and Purification Problems of RhoGDI Using a Family of “Parallel” Expression Vectors

Cited by 615 publications

References 20 publications

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain

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