Johnson T M, Holaday S K, Sun Y, Subramaniam P S, Johnson H M, Krishna N R
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294, USA.
J Interferon Cytokine Res. 1999 Jun;19(6):631-6. doi: 10.1089/107999099313776.
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.
干扰素 -τ（IFN-τ）是一种新型的I型干扰素，最初被鉴定为妊娠识别激素。IFN-τ具有其他I型干扰素的所有生物学特性，包括通过诱导细胞周期抑制剂基因产物p21WAF1发挥抗病毒活性和抗增殖活性。它是治疗癌症、病毒感染以及自身免疫性疾病（如多发性硬化症）的一种有前景的疗法，且没有与IFN-α和IFN-β相关的不良副作用。在此，我们描述了在用于溶液中初始二维和三维核磁共振研究的基本培养基中，从毕赤酵母表达功能活性且均匀15N标记的IFN-τ的新生长和诱导条件。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离飞行时间质谱仪（MALDI-TOF MS）测定，纯化的15N-IFN-τ是均一的，并且保留了完整的生物学活性。质谱分析证实IFN-τ的15N掺入均匀，掺入率超过99%。圆二色性（CD）以及一维核磁共振和15N-1H异核单量子相干（HSQC）光谱证实纯化的15N标记的IFN-τ具有稳定的二级结构。除了为IFN-τ的同位素标记提供一条途径外，我们的方法可能对在基本培养基中生长的毕赤酵母中难以获得的其他蛋白质的表达和纯化有用。
