Ti Z C, Gooley A A, Slade M B, Bowers V M, Williams K L
School of Biological Sciences, Macquarie University, Sydney, Australia.
J Biotechnol. 1990 Nov;16(3-4):233-43. doi: 10.1016/0168-1656(90)90039-e.
Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.
本文描述了对一种盘基网柄菌细胞表面糖蛋白的大规模纯化，该糖蛋白通过糖基磷脂酰肌醇（GPI）部分锚定在膜上。纯化方案包括四个步骤：通过低速离心分离粗细胞膜，用丙酮去除这些膜的脂质，用去污剂β-D-硫代葡萄糖苷辛酯（OTP）提取膜蛋白，以及通过单克隆抗体免疫亲和色谱法纯化特定的膜蛋白。纯化得到的蛋白PsA（前孢子特异性抗原）是一种在发育过程中受调控的膜糖蛋白，存在于细胞黏菌盘基网柄菌的一部分细胞上。该方案提供了一种高效、经济且技术简单的方法，能够大量纯化GPI蛋白以用于结构和功能研究。PsA的回收率约为60%；纯度为97%，提取1×10¹⁰个细胞（干重1.1克）可得到约0.5毫克PsA糖蛋白。文中还介绍了在不锈钢托盘中低成本培养千克级盘基网柄菌细胞的技术。盘基网柄菌作为生产外源膜相关蛋白的新型表达系统具有巨大潜力。该纯化策略提供了一种纯化其他GPI蛋白的方法，包括那些通过蛋白质工程技术生产的蛋白。
