Witzgall, Ralph and O'Leary, Eileen and Bonventre, Joseph V.
A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail.
Analytical Biochemistry 223 (2), pp. 291-298.
Expression of newly cloned cDNAs in mammalian cell lines is an essential tool for the functional analysis of the proteins encoded by these cDNAs. In many instances, however, evaluation of the protein is difficult because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins on Western blots or for immunocytochemistry or immunoprecipitation. A number of gene fusion systems have been employed in which a known peptide is fused to the expression product of interest and the fusion protein is purified using affinity chromatography and identified in extracts or by immunocytochemistry using antibodies directed against the affinity handle peptide. The DNA-binding domain of the yeast transcription factor GAL4 is widely used to construct fusion proteins with putative transcription factors to evaluate potential trans-acting domains. Because of the lack of commercially available anti-GAL4 antibodies, the further biochemical characterization of these fusion proteins has remained difficult. We describe the construction of two mammalian expression vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for pM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast transcription factor GAL4 with a Flag peptide (consisting of the 11-amino-acid leader peptide of the gene 10 product from bacteriophage T7) at the NH2-terminus and a tail of six histidines at the COOH-terminus. Unique restriction sites allow both the construction of fusion proteins with the GAL4 DNA-binding domain and the replacement of the GAL4 fragment with another insert. Proteins encoded by this vector are biologically active, can be easily precipitated and purified by interaction of its histidine tail with immobilized Ni2+, and can be visualized on Western blots with an antibody against the Flag peptide.