Sørensen Steen, Justesen Steen Just, Johnsen Anders H
Department of Clinical Biochemistry, Hvidovre Hospital, Kettegaard Alle 30, 2650 Hvidovre, Denmark.
Protein Expr Purif. 2003 Aug;30(2):238-45. doi: 10.1016/s1046-5928(03)00102-5.
Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.
骨桥蛋白（OPN）在许多器官和组织中表达，并且就大小和翻译后修饰而言，与不同分子形式相关的生物学特性也有所不同。然而，目前尚缺乏从可获取的生物来源中纯化出真实完整的OPN及其片段的方法。基于人乳中OPN的晶体生长抑制活性，采用了一种四步程序从人乳中纯化OPN，该程序包括阴离子交换色谱法、酪蛋白去除、羟基磷灰石色谱法和负亲和色谱法。通过两步程序，即尺寸排阻色谱法和反相色谱法，将纯化后的OPN进一步分离为不同的分子形式。用纯化后的完整OPN和高分子量OPN组分制备了兔多克隆抗体；通过酶免疫测定法（EIA）检测时，两种形式的免疫反应性几乎相等。这些程序有助于纯化完整的OPN和OPN片段，以用于标准化、单特异性抗体的制备及功能研究。
