Species: Saccharomyces cerevisiae and Human herpesvirus 2
Expression Host: E. coli
Tag: His-tag
Purity: 90%
Molecular Weight: 27.8 kDa.
Gene Accession Number: NM_001184062 + M60050.
Purification and Quality Control
The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified GAL4-VP16 is greater than 90% homogeneous based on SDS-PAGE analysis.
Unit Definition (Activity)
1 unit equals 1 nanogram of purified protein. 20 unit is sufficient for a gel mobility shift assay in a 20 µl reaction.
Applications
GAL4-VP16 has been applied in in vitro transcription assays and protein-protein interactions assays.
Formulation and Storage
The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.
Background
The GAL4 protein of yeast activates the transcription of several genes involved in galactose metabolism. This event requires that GAL4 bind to upstream activation sites with the consensus sequence 5`-CGGN5(T/A)N5CCG-3` (1 ). A fragment of the GAL4 protein, comprising amino acids 1-147, binds DNA but fails to activate transcription (2). Herpes virus VP16 activates expression of immediate early genes in virally-infected cells (3). As most other eukaryotic transcriptional activator proteins, VP16 has a modular domain structure: it’s N-terminus is involved in DNA-protein interactions, while it’s C-terminal 79 amino acids have proven to be an especially potent transactivation domain (4). When fused to the DNA-binding domain of the yeast GAL4, this VP16 fragment functions as an activator of transcription in yeast, mammalian cells and in vitro transcription assays (5, 6). VP16 has been shown to bind to TBP (7), TFIIB (8), and replication factor A (9). Recombinant GAL4-VP16 is isolated from an E. coli strain that carries the coding sequence of the fused protein under the control of a T7 promoter.
References
1. Kodadek T. (1993) Cell Mol Biol Res. 39, 355-360 2. Keegan, L., et al., (1986) Science 231, 699-704 3. Post, L.E., et al., (1981) Cell 24, 555-565 4. Triezenberg, S.J., et al., (1988) Genes Dev. 2, 718-729 5. Sadowski, I., et al., (1988) Nature 335, 563-564 6. Cousens, D.J., et al., (1989) EMBO J. 8, 2337-2342 7. Stringer, K. F., et al., (1990) Nature 345, 783-786 8. Lin, Y-S., et al., (1991) Nature 353, 569-571 9. He, Z., et al., (1993) 73, 1223-1232
DISCLAIMER
This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.
