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-Feb-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
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