Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies

Cole, Taylor R.; Igumenova, Tatyana I.

doi:10.1016/j.pep.2014.12.018

Cited by 6 publications

(4 citation statements)

References 35 publications

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“…The GST fusion system has been used extensively as a prokaryotic expression system in bacteria for its convenience at producing soluble heterologous proteins that are easy to purify [ 30 ]. Here, GST was employed as a co-expression vehicle with C-TgNHE1, with the aim to generate a soluble functional fusion protein.…”

Section: Discussionmentioning

confidence: 99%

A Novel Polyclonal Antiserum against <i>Toxoplasma gondii</i> Sodium Hydrogen Exchanger 1

et al. 2016

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The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

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Section: Discussionmentioning

confidence: 99%

A Novel Polyclonal Antiserum against <i>Toxoplasma gondii</i> Sodium Hydrogen Exchanger 1

et al. 2016

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“…Knock-in cell line creation and sorting:A ll synthetic nucleic acid reagents were purchased from IDT.s gRNAs and Cas9/sgRNA ribonucleoprotein (RNP) complexes were prepared as described previously. [15] In order to simplify the isolation of integrated cells, we introduced GFP 11 (16 residues) [16] in tandem with SpyTag into cells stably expressing GFP [1][2][3][4][5][6][7][8][9][10] .C ells were then sorted with fluorescenceactivated cell sorting (FACS) by using the GFP signal. We note that in practical applications, GFP can be omitted, and SpyTag-positive cells can be identified through the classic clonal selection method.…”

Section: Methodsmentioning

confidence: 99%

“…Epitope tags, short peptides that can be recognized by antibodies, are widely used in fluorescent labeling of fixed-cell proteins form icroscopy analysis, especially in numerous cases in which high-quality antibodies directly against the target protein are unavailable. Because of the small size of epitope tags, their use in labeling proteins is less likely to perturb fusion protein function or structuralo rganization compared to using fluorescent proteins or enzymatic tags such as SNAP-tag [1] and Halo-tag. [2] With recent advancements in genome editing technologies, this smalls ize also facilitatess ystematic labeling of endogenous genes.…”

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confidence: 99%

Covalent protein labeling by SpyTag-SpyCatcher in fixed cells for super-resolution microscopy

Pessino

Citron

Feng

2017

Preprint

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Labeling proteins with high specificity and efficiency is afundamental prerequisite for microscopic visualization of subcellular protein structuresa nd interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins,t hey require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescentl abeling of intracellular proteins in fixed cells for both conventionala nd super-resolutionm icroscopy. We also appliedt his method to endogenous proteins by gene editing, demonstratingi ts high labeling efficiency and capability for isoform-specific labeling.

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“…To determine the effect of divalent metal ions on the N-term regulatory region of PKC, we conducted SAXS experiments (sections S1−3 and Table S2) on the 22.3 kDa C1B−C2 region from PKCα and the 30.6 kDa chimeric full regulatory region C1C2c that additionally contains the C1A domain from the γ isoform. 23 Three metal ions were used: Cd 2+ , Pb 2+ , and native activator Ca 2+ . Unexpectedly, we found that Cd 2+ and Pb 2+ caused significant changes in protein morphology compared to Ca 2+ (Figure 2A).…”

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confidence: 99%

Cd(II)- and Pb(II)-Induced Self-Assembly of Peripheral Membrane Domains from Protein Kinase C

et al. 2018

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Cd 2+ and Pb 2+ are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd 2+ and Pb 2+ causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca 2+ -binding motifs of protein kinase C. The ability of protein oligomers to interact with anionic membranes is enhanced compared to the monomeric species. Our findings suggest that metal-ion-dependent peripheral membrane domains can be utilized for generating protein−metal-ion nanoclusters and serve as biotemplates for the design of sequestration agents. Communication pubs.acs.org/biochemistry

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Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies

Cited by 6 publications

References 35 publications

A Novel Polyclonal Antiserum against <i>Toxoplasma gondii</i> Sodium Hydrogen Exchanger 1

A Novel Polyclonal Antiserum against <i>Toxoplasma gondii</i> Sodium Hydrogen Exchanger 1

Covalent protein labeling by SpyTag-SpyCatcher in fixed cells for super-resolution microscopy

Cd(II)- and Pb(II)-Induced Self-Assembly of Peripheral Membrane Domains from Protein Kinase C

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