A versatile expression vector for the in vitro study of protein-protein interactions: characterization of E47 mutant proteins.
Several mutants of the E47 protein, a member of the family of basic/helix-loop-helix (b-HLH) transcriptional regulators, were examined for their ability to homo- and heterodimerize with the protein product of the T-cell oncogene tal-1/SCL. For this purpose, a novel bacterial expression system was developed in which proteins are expressed as fusions appended to glutathione-S-transferase via a thrombin cleavage site and either one or four protein kinase recognition sites embedded in a glycine-rich domain. Since the interaction domain can be purified away from the glutathione-S-transferase moiety and the radioactive label is located in a flexible N-terminal tag, protein folding should occur normally. Our studies with E47 proteins prepared in this system indicate that the ratio between E47 homodimers and E47/tal-1 heterodimers can dramatically shift upon subtle mutations in the loop region and the second helix of the E47 protein. This unexpected results suggests a novel mechanism to alter the equilibrium between different transactivating protein complexes of the b-HLH class.
Hainzl
T
T
Department of Medicine I, University of Freiburg, Germany.
Boehm
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C544912:TCF7L1 protein, human; C067143:TAL1 protein, human