標題: | Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography |
作者: | Tseng, CF Huang, HY Yang, YT Mao, SJT 生物科技學系 Department of Biological Science and Technology |
關鍵字: | human haptoglobin 1-1;2-1;2-2;affinity purification;alpha-helix;monoclonal antibodies |
公開日期: | 1-二月-2004 |
摘要: | Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp, phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed. (C) 2003 Elsevier Inc. All rights reserved. |
URI: | http://dx.doi.org/10.1016/j.pep.2003.09.006 http://hdl.handle.net/11536/148612 |
ISSN: | 1046-5928 |
DOI: | 10.1016/j.pep.2003.09.006 |
期刊: | PROTEIN EXPRESSION AND PURIFICATION |
Volume: | 33 |
起始頁: | 265 |
結束頁: | 273 |
顯示於類別: | 期刊論文 |