pCold-GST vector: A novel cold-shock vector containing GST tag for soluble protein production

Hayashi, Kokoro; Kojima, Chojiro

doi:10.1016/j.pep.2008.07.007

Cited by 75 publications

(51 citation statements)

References 13 publications

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“…DCX cDNA was amplified by PCR using primers 5Ј-GAAGATCTATGGAACTTGATTTTGGACAT-TTTG-3Ј and 5Ј-CTTCAAGCTTTCACATGGAATCGCC-AAG-3Ј. Full-length DCX cDNA was inserted into plasmids including pCMV5 containing an N-terminal FLAG tag, pmCherry-C1 (Clontech), pEGFP-C1 (Clontech), and the pCold-GST vector (27), which was generously provided by Dr. Kojima (Osaka University). Point mutants of DCX at Ser-47 were generated using a QuikChange II site-directed mutagenesis kit (Stratagene) and the following primers: S47A, 5Ј-GAACCTT-GCAGGCATTAGCTAATGAGAAGAAGGCCAAG-3Ј; S47E, 5Ј-GAACCTTGCAGGCATTAGAGAATGAGAAGAAGGCC-AAG-3Ј; and S47R, 5Ј-GAACCTTGCAGGCATTAAGA-AATGAGAAGAAGGCCAAG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Purification of Recombinant Proteins-To express recombinant proteins of wild-type and mutant DCX, their cDNAs were inserted into modified pCold-I-GST plasmids (27). Protein expression of His-GST-tagged DCX in the BL21 (DE3) codon plus strain was induced by 0.1 mM IPTG at 16°C for 24 h. Proteins were extracted in PBS with sonication.…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of Doublecortin by Protein Kinase A Orchestrates Microtubule and Actin Dynamics to Promote Neuronal Progenitor Cell Migration

Toriyama¹,

Mizuno²,

Fukami

et al. 2012

Journal of Biological Chemistry

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Background: Regulation of neuronal progenitor cell migration is critical for proper brain lamination. Results: G protein-coupled receptor signaling promotes cell migration, lamellipodium formation, and Rac activation by phosphorylating a microtubule-associated protein, doublecortin. Conclusion: Doublecortin is released from microtubules and induces actin reorganization in a phosphorylation-dependent manner. Significance: This is the first evidence for the coordinated regulation of microtubule and actin dynamics.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Doublecortin by Protein Kinase A Orchestrates Microtubule and Actin Dynamics to Promote Neuronal Progenitor Cell Migration

Toriyama¹,

Mizuno²,

Fukami

et al. 2012

Journal of Biological Chemistry

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“…Purification of Recombinant Proteins-The cDNA of B-Ras2 and T18N mutant was cloned into pCold-I-GST vector, with GST tag inserted into the original pCold-I bacterial expression vector (Takara, Shiga, Japan). pCold-I-GST vector was a gift from Dr. Chojiro-Kojima of Nara Institute of Science and Technology (45). The cDNA of p65/RelA, p65/RelA (S276A), and IB␣ was cloned into pCold-I expression vector.…”

Section: Methodsmentioning

confidence: 99%

κB-Ras Is a Nuclear-Cytoplasmic Small GTPase That Inhibits NF-κB Activation through the Suppression of Transcriptional Activation of p65/RelA

Tago

Funakoshi‐Tago

Sakinawa

et al. 2010

Journal of Biological Chemistry

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NF-B is an important transcription factor involved in various biological responses, including inflammation, cell differentiation, and tumorigenesis. B-Ras was identified as an IB-interacting small GTPase and is reported to disturb cytokine-induced NF-B activation. In this study, we established that B-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDPbinding form of the B-Ras mutant exhibited a more potent inhibitory effect on NF-B activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/ RelA NF-B subunit. B-Ras suppressed phosphorylation at serine 276 on the p65/RelA subunit, resulting in decreased interaction between p65/RelA and the transcriptional coactivator p300. Interestingly, the GDP-bound B-Ras mutant exhibited higher interactive affinity with p65/RelA and inhibited the phosphorylation of p65/RelA more potently than wild-type B-Ras. Taken together, these findings suggest that the GDPbound form of B-Ras in cytoplasm suppresses NF-B activation by inhibiting its transcriptional activation.

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“…The vinculin P1 mutant (P843A, P844A, P846A, P847A), P2 mutant (P860A, P861A, P863A, P864A) and P3 mutant (P872A, P873A, P875A, P876A, P877A) were generated by using the QuikChange Site-Directed Mutagenesis Kit [Agilent Technologies (Stratagene), Santa Clara, CA]. Vinculin tail probes were subcloned into pColdI vector (Takara Bio, Ohtsu, Japan) for purification, and vinexin aDN, which lacks the Nterminal 156 amino acids of vinexin a, was subcloned into the pColdI-GST vector (kindly provided by Chojiro Kojima) (Hayashi and Kojima, 2008). A plasmid encoding GST-VASP (Maruoka et al, 2012) was a gift from Masahiro Maruoka.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Interaction of the vinculin proline-rich linker region with vinexin α in sensing extracellular matrix stiffness

Yamashita

Ichikawa

Matsuyama

et al. 2014

Journal of Cell Science

125

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BSTRACTAlthough extracellular matrix (ECM) stiffness is an important aspect of the extracellular microenvironment and is known to direct the lineage specification of stem cells and affect cancer progression, the molecular mechanisms that sense ECM stiffness have not yet been elucidated. In this study, we show that the proline-rich linker (PRL) region of vinculin and the PRL-region-binding protein vinexin are involved in sensing the stiffness of ECM substrates. A rigid substrate increases the level of cytoskeleton-associated vinculin, and the fraction of vinculin stably localizing at focal adhesions (FAs) is larger on rigid ECM than on soft ECM. Mutations in the PRL region or the depletion of vinexin expression impair these responses to ECM stiffness. Furthermore, vinexin depletion impairs the stiffness-dependent regulation of cell migration. These results suggest that the interaction of the PRL region of vinculin with vinexin a plays a crucial role in sensing ECM stiffness and in mechanotransduction.

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pCold-GST vector: A novel cold-shock vector containing GST tag for soluble protein production

Cited by 75 publications

References 13 publications

Phosphorylation of Doublecortin by Protein Kinase A Orchestrates Microtubule and Actin Dynamics to Promote Neuronal Progenitor Cell Migration

Phosphorylation of Doublecortin by Protein Kinase A Orchestrates Microtubule and Actin Dynamics to Promote Neuronal Progenitor Cell Migration

κB-Ras Is a Nuclear-Cytoplasmic Small GTPase That Inhibits NF-κB Activation through the Suppression of Transcriptional Activation of p65/RelA

Interaction of the vinculin proline-rich linker region with vinexin α in sensing extracellular matrix stiffness

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