[6] Quantitation of protein

Stoscheck, Christa M.

doi:10.1016/0076-6879(90)82008-p

Cited by 1,307 publications

(781 citation statements)

References 36 publications

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“…Many procedures exist for protein quantification, with all having their specific advantages and limitations. 31,32 Colorimetric protein quantification assays, including the Bradford and Lowry methods, are frequently used due to their sensitivity and quick procedures. 31,33 However, accuracy of colorimetric methods depends on the protein and these methods require standards for obtaining actual concentration values.…”

Section: Discussionmentioning

confidence: 99%

“…31,33 However, accuracy of colorimetric methods depends on the protein and these methods require standards for obtaining actual concentration values. 31,32 The PHA method is a colorimetric technique for quantifying proteins containing intrinsic heme and is the most used method for this purpose. 34 We have shown here that the purification of proteins using the heme-tag-HIS method offers the additional advantage in that the proteins can be easily quantified using the PHA.…”

Section: Discussionmentioning

confidence: 99%

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A heme fusion tag for protein affinity purification and quantification

Asher

Bren

2010

Protein Science

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We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200-500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A heme fusion tag for protein affinity purification and quantification

Asher

Bren

2010

Protein Science

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“…Protein was concentrated to 0.5-1.0 mg ml −1 and dialyzed against storage buffer (50 mM HEPES, pH 7.5, 250 mM NH 4 Cl, 1 mM EDTA, 10% (w/v) glycerol). Concentrations of protein and any residual RNA were assessed by the Warburg-Christian method 51 . Purity was assessed by SDS-PAGE.…”

Section: Hfq Expression and Purificationmentioning

confidence: 99%

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Mikulecky

Kaw

Brescia

et al. 2004

Nat Struct Mol Biol

226

395

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The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in posttranscriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.Hfq protein from Escherichia coli was first described in connection with Qβ-phage replication 1,2 . Hfq has recently emerged as a central player in post-transcriptional gene regulation as mediated by bacterial ncRNAs [3][4][5][6] . Escherichia coli Hfq mutants show disrupted signaling in stress response pathways 7,8 , arising from the need for Hfq to mediate base-pairing between regulatory ncRNAs and their mRNA targets. Examples of these partnerships include DsrA-rpoS 7,9,10 , OxyS-fhlA 11,12 , OxyS-rpoS 13 , RprA-rpoS 14 , RyhBsodB [15][16][17] .Complexes between ncRNAs and their mRNA targets function in several ways. Most commonly, complexed structures lead to translational activation or repression by remodeling mRNA regulatory regions containing the ribosome-binding site (RBS) and/or start codon. Alternatively, the interaction can enhance decay of the target mRNA16 or simply block translation11. Clearly, Hfq facilitates base-pairing between ncRNAs and their targets, but how it does so is poorly understood. How the chaperone function relates to other Hfq activities such as the control of poly(A) tail elongation19 , 20 and regulation of mRNA stability21 , 22 is also unknown. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2011 April 5. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHfq shares sequence similarity to the eukaryotic Lsm proteins [23][24][25][26][27] We addressed these questions through a mutational analysis of Hfq, probing in vitro binding to several model RNAs that represent species with which Hfq must interact. Hfq mutants were assayed in vivo using a reporter assay and RNA lifetime experiments. Together, the results support a model wherein at least two independent RNA-binding sites exist on the Hfq hexamer, and juxtaposition of bound RNAs facilitates base-pairing. RESULTS Hfq mutagenesisTo identify amino acids essential for RNA binding, we constructed a series of E. coli Hfq misse...

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“…Protein concentrations were determined using the Protein Assay Dye Reagent Concentrate (Bio-Rad) following treatment of the samples with NaOH as described previously (28). Briefly, protein samples were treated with 0.5 M NaOH according to the method of Stoscheck (29) in 60 μL for 10 min at room temperature. Then 1 mL of the diluted dye reagent (1:5 in water) was added to each sample.…”

Section: Expression Of Ttp In Transfected Humanmentioning

confidence: 99%

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Cao

2004

Biochemistry

122

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Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AUrich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor α mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 μM Zn 2+ . The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zincdependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex. Tristetraprolin (TTP)1 (also known as ZFP36, Nup475, TIS11, and G0S24) is a prototypical member of a small family of mammalian proteins with tandem CCCH (CX 8 CX 5 CX 3 H) zinc finger motifs separated by 18 amino acid residues (1). Similar zinc finger sequences are found in at least 19 species in the GenBank database, including human, cow, mouse, rat, sheep,

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[6] Quantitation of protein

Cited by 1,307 publications

References 36 publications

A heme fusion tag for protein affinity purification and quantification

A heme fusion tag for protein affinity purification and quantification

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

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[6] Quantitation of protein

Cited by 1,307 publications

References 36 publications

A heme fusion tag for protein affinity purification and quantification

A heme fusion tag for protein affinity purification and quantification

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

Contact Info

Product

Resources

About

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,