A MAMMALIAN EXPRESSION VECTOR FOR THE EXPRESSION OF GAL4 FUSION PROTEINS WITH AN EPITOPE TAG AND HISTIDINE TAIL

Expression of newly cloned cDNAs in mammalian cell lines is an essenti al tool for the functional analysis of the proteins encoded by these c DNAs. In many instances, however, evaluation of the protein is difficu lt because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins o n Western blots or for immunocytochemistry or immunoprecipitation. A n umber of gene fusion systems have been employed in which a known pepti de is fused to the expression product of interest and the fusion prote in is purified using affinity chromatography and identified in extract s or by immunocytochemistry using antibodies directed against the affi nity 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. Bec ause of the lack of commercially available anti-GAL4 antibodies, the f urther biochemical characterization of these fusion proteins has remai ned difficult. We describe the construction of two mammalian expressio n vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for gM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast tran scription factor GAL4 with a Flag peptide (consisting of the 11-aminoa cid leader peptide of the gene 10 product from bacteriophage T7) at th e NH2-terminus and a tail of six histidines at the COOH-terminus. Uniq ue restriction sites allow both the construction of fusion proteins wi th the GAL4 DNA-binding domain and the replacement of the GAL4 fragmen t with another insert. Proteins encoded by this vector are biologicall y active, can be easily precipitated and purified by interaction of it s histidine tail with immobilized Ni2+, and can be visualized on Weste rn blots with an antibody against the Flag peptide. (C) 1994 Academic Press, Inc.