title: Expression, fermentation and purification of a predicted intrinsically disordered region of the transcription factor, NFAT5.
abstract: Hypertonicity stimulates Nuclear Factor of Activated T-cells 5 (NFAT5) nuclear localization and transactivating activity. Many transcription factors are known to contain intrinsically disordered regions (IDRs) which become more structured with local environmental changes such as osmolality, temperature and tonicity. The transactivating domain of NFAT5 is predicted to be intrinsically disordered under normal tonicity, and under high NaCl, the activity of this domain is increased. To study the binding of co-regulatory proteins at IDRs a cDNA construct expressing the NFAT5 TAD was created and transformed into Escherichia coli cells. Transformed E. coli cells were mass produced by fermentation and extracted by cell lysis to release the NFAT5 TAD. The NFAT5 TAD was subsequently purified using a His-tag column, cation exchange chromatography as well as hydrophobic interaction chromatography and then characterized by mass spectrometry (MS).
authors.affiliation: National Institutes of Health, National Heart, Lung and Blood Institute, Systems Biology Center, Bethesda, MD 20892, United States. Electronic address: jenna.dumond@nih.gov.
authors.forename: Jenna F
authors.initials: JF
authors.lastname: DuMond
authors.affiliation: National Institutes of Health, National Heart, Lung and Blood Institute, Biochemistry and Biophysics Center, Bethesda, MD 20892, United States. Electronic address: hey4@nhlbi.nih.gov.
authors.forename: Yi
authors.initials: Y
authors.lastname: He
authors.affiliation: National Institutes of Health, National Heart, Lung and Blood Institute, Systems Biology Center, Bethesda, MD 20892, United States. Electronic address: burgm@mail.nih.gov.
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
authors.affiliation: National Institutes of Health, National Heart, Lung and Blood Institute, Systems Biology Center, Bethesda, MD 20892, United States. Electronic address: ferraris@nhlbi.nih.gov.
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D064267:Intrinsically Disordered Proteins; C118125:NFAT5 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors
keywords: Fermentation; Hypertonicity; Intrinsically disordered proteins; Transcription factors