Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris.
The metal response element binding transcription factor-1 (MTF-1) is an important stress response, heavy metal detoxification, and zinc homeostasis factor in eukaryotic organisms from Drosophila to humans. MTF-1 transcriptional regulation is primarily mediated by elevated levels of labile zinc, which direct MTF-1 to bind the metal response element (MRE). This process involves direct zinc binding to the MTF-1 zinc fingers, and zinc dependent interaction of the MTF-1 acidic region with the p300 coactivator protein. Here, the first recombinant expression system for mutant and wild type (WT) mouse MTF-1 (mMTF-1) suitable for biochemical and biophysical studies in vitro is reported. Using the methyltropic yeast Pichia pastoris, nearly half-milligram recombinant WT and mutant mMTF-1 were produced per liter of P. pastoris cell culture, and purified by a FLAG-tag epitope. Using a first pass ammonium sulfate purification, followed by anti-FLAG affinity resin, mMTF-1 was purified to >95% purity. This recombinant mMTF-1 was then assayed for direct protein-protein interactions with p300 by co-immunoprecipitation. Surface plasmon resonance studies on mMTF-1 provided the first quantitative DNA binding affinity measurements to the MRE promotor element (K(d)=5±3 nM). Both assays demonstrated the functional activity of the recombinant mMTF-1, while elucidating the molecular basis for mMTF-1-p300 functional synergy, and provided new insights into the mMTF-1 domain specific roles in DNA binding. Overall, this production system provides accessibility for the first time to a multitude of in vitro studies using recombinant mutant and WT mMTF-1, which greatly facilitates new approaches to understanding the complex and varied functions of this protein.
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, United States. rwhuyck@mail.umkc.edu
Ryan W
RW
Huyck
Andrew
A
Keightley
John H
JH
Laity
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D050881:E1A-Associated p300 Protein / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D060066:Protein Interaction Maps; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000097809:Transcription Factor MTF-1
D004247:DNA; D004268:DNA-Binding Proteins; D050881:E1A-Associated p300 Protein; D011994:Recombinant Proteins; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1; C494361:Ep300 protein, mouse