Expression and Reconstitution of NF-κB from Insect Cells Using a Baculovirus Vector

Coleman, Timothy A.; Huddleston, Kathleen A.; Ruben, Steven M.; Rosen, Craig A.; Gentz, Reiner

doi:10.1006/prep.1996.0670

Cited by 6 publications

(4 citation statements)

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“…The putative protein coding sequence was then ligated into the baculovirus protein expression vector, pA2 (15), and recombinant protein was expressed and purified. The purified recombinant neuroserpin migrated as a doublet with relative molecular masses of 49 and 47 kDa on SDS-PAGE in close agreement with the predicted molecular mass of 44 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…Expression and Purification of Recombinant Neuroserpin in Insect Cells-The protein-coding region of neuroserpin was amplified from a full-length neuroserpin cDNA with Pfu polymerase, and the product was ligated to the BamHI and XbaI sites of the pA2 baculovirus expression vector (15). Following transformation into DH5␣ Escherichia coli, plasmid DNA was isolated, and the entire coding region sequenced to exclude any polymerase chain reaction generated errors.…”

Section: Methodsmentioning

confidence: 99%

“…Following transformation into DH5␣ Escherichia coli, plasmid DNA was isolated, and the entire coding region sequenced to exclude any polymerase chain reaction generated errors. Plasmid DNA was then purified and transfected into Sf9 insect cells to generate a recombinant virus as described previously (15). For protein production, Sf9 cells were seeded in serum-free media at a density of 1.5 ϫ 10 6 cells/ml in spinner flasks.…”

Section: Methodsmentioning

confidence: 99%

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Neuroserpin, a Brain-associated Inhibitor of Tissue Plasminogen Activator Is Localized Primarily in Neurons

Hastings¹,

Coleman²,

Haudenschild³

et al. 1997

Journal of Biological Chemistry

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197

212

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A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6.2 ؋ 10 5 M ؊1 s ؊1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-␥ but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Neuroserpin, a Brain-associated Inhibitor of Tissue Plasminogen Activator Is Localized Primarily in Neurons

Hastings¹,

Coleman²,

Haudenschild³

et al. 1997

Journal of Biological Chemistry

Self Cite

197

212

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“…Having performed selections using truncated recombinant p50 2 (amino acids 40-366), we addressed the ability of RNA aptamer 7 to bind to a full-length recombinant form of p50 2 (amino acids 1-504), as well as to a recombinant form of the transcriptionally active heterodimer composed of p50/ p65 subunits. These proteins were expressed in a baculovirus system (32), and RNA aptamer binding was assessed in gel shift competition assays (Figure 6). As previously observed for p50 2 , RNA aptamer 7 competed with radiolabeled DNA duplex 1 for binding to full-length p50 2 (Figure 6A), whereas only modest competition was seen with unlabeled DNA duplex 2 or RNA 6 (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

Selection and Characterization of an RNA Decoy for Transcription Factor NF-κB

1999

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Despite their chemical similarity, DNA and RNA sequences typically adopt very different structures within cells and are recognized by different proteins. However, a few interesting examples of proteins with dual specificity for DNA and RNA have previously been noted. These observations raise the possibility that RNA surrogates might be identified for many transcription factors that normally bind DNA. As an initial test of this novel concept, we used in vitro selection to isolate a small RNA aptamer that binds with nanomolar affinity to human transcription factor NF-kappa B, a key regulator of inflammation, HIV-1 gene expression, and apoptosis. Selected RNAs contain a 31-nucleotide core domain that was shown by mutation and deletion analyses to be necessary and sufficient for NF-kappa B binding. Neither DNA nor 2'-O-methyl RNA analogues of the aptamer bound NF-kappa B. The results of competition experiments demonstrate that binding of the RNA aptamer blocks the ability of NF-kappa B to bind duplex DNA. Expression of this aptamer structure within heterologous nuclear RNA transcripts may provide a new strategy to inhibit NF-kappa B function in vivo. Aptamers that inhibit transcription factors might be useful in a variety of applications.

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Functional Characterization of a Purified, Recombinant NF-κB/IκB Complex

Larson

Mercurio

et al. 1999

Biochemical and Biophysical Research Communications

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Expression and Reconstitution of NF-κB from Insect Cells Using a Baculovirus Vector

Cited by 6 publications

References 0 publications

Neuroserpin, a Brain-associated Inhibitor of Tissue Plasminogen Activator Is Localized Primarily in Neurons

Neuroserpin, a Brain-associated Inhibitor of Tissue Plasminogen Activator Is Localized Primarily in Neurons

Selection and Characterization of an RNA Decoy for Transcription Factor NF-κB

Functional Characterization of a Purified, Recombinant NF-κB/IκB Complex

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