Expression, purification, and characterization of interferon-tau produced in Pichia pastoris grown in a minimal medium.
Interferon-tau (IFN-tau) is a novel type I IFN that was originally identified as a pregnancy recognition hormone. IFN-tau shares all of the biological properties of other type I IFNs including antiviral activity and antiproliferative activity through induction of the cell cycle inhibitor gene product p21WAF1. It is a promising therapy for cancers, viral infections, and for autoimmune disorders such as multiple sclerosis, without the adverse side effects associated with IFN-alpha and IFN-beta. Here, we describe novel growth and induction conditions for the expression of functionally active and uniformly 15N-labeled IFN-tau from Pichia pastoris in a minimal media for use in initial 2D- and 3D-NMR studies in solution. Purified 15N-IFN-tau was homogenous, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometer (MS), and retained full biological activity. MS analysis confirmed uniform isotopic labeling of IFN-tau with 15N incorporation exceeding 99%. Circular dichroism (CD) as well as 1D-NMR and 15N-1H heteronuclear single quantum coherence (HSQC) spectra confirmed that purified 15N-labeled IFN-tau has a stable secondary structure. Besides providing a route for isotope labeling of IFN-tau, our procedure may be useful for the expression and purification of other proteins that are difficult to obtain in Pichia pastoris grown in minimal media.
Johnson
T M
TM
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294, USA.
Holaday
S K
SK
Sun
Y
Y
Subramaniam
P S
PS
Johnson
H M
HM
Krishna
N R
NR
D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D005260:Female; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010843:Pichia / Q000187:drug effects* / Q000254:growth & development; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000998:Antiviral Agents; D003470:Culture Media; D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau