Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies
Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies
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DOI：
10.1006/prep.2001.1594
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发表时间：
2002-04-01
影响因子：
1.6
通讯作者：
Steinkasserer, A
中科院分区：
文献类型：
--
作者：
Lechmann, M;Kremmer, E;Steinkasserer, A
CD 83是一种分子量为45-kDa的糖蛋白，属于免疫球蛋白（IG）超家族成员，是成熟树突状细胞最著名的标志物。虽然CD 83的确切功能尚不清楚，但其与共刺激分子CD 80和CD 86一起的选择性表达和上调表明CD 83在诱导免疫应答中的重要作用。为了进行功能研究并阐明其作用模式，获得重组表达和高度纯化的CD 83分子至关重要。因此，将人CD 83的外部IG结构域（hCD 83 ext）表达为GST融合蛋白（GST-hCD 83 ext），并在天然条件下纯化可溶性蛋白。融合蛋白用GSTrap柱纯化，然后用阴离子交换层析。然后使用凝血酶切割GSTh ⑶ 83 ext，并使用GSTrap柱进一步纯化可溶性hCD 83 ext，最后通过制备型凝胶过滤作为精制步骤，并用于进一步表征。纯化的GST-hCD 83融合蛋白也用于在大鼠系统中产生单克隆抗CD 83抗体。产生了两种不同的单克隆抗体。使用这些抗体，在FACS和Western印迹分析中特异性识别CD 83。此外，我们表明天然CD 83是糖基化的，并且这种糖基化影响蛋白质印迹分析中抗体的结合。最后，纯化的hCD 83 ext蛋白通过一维NIM分析，这些分析强烈表明hCD 83 ext是折叠的，因此可以用于进一步的结构和功能研究。(C)2002 Elsevier Science（美国）。
CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily, It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GSThCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NI M and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies. (C) 2002 Elsevier Science (USA).