Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris
Protein Expression and Purification, ISSN: 1046-5928, Vol: 85, Issue: 1, Page: 86-93
2012
- 5Citations
- 21Captures
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Metrics Details
- Citations5
- Citation Indexes5
- CrossRef5
- Captures21
- Readers21
- 21
Article Description
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.
Bibliographic Details
http://www.sciencedirect.com/science/article/pii/S104659281200188X; http://dx.doi.org/10.1016/j.pep.2012.06.018; http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84864402383&origin=inward; http://www.ncbi.nlm.nih.gov/pubmed/22780964; https://linkinghub.elsevier.com/retrieve/pii/S104659281200188X; https://dx.doi.org/10.1016/j.pep.2012.06.018
Elsevier BV
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