重组DNA
血红蛋白
生物
分子生物学
珠蛋白
表达式向量
亲和层析
变构调节
融合蛋白
生物化学
基因
酶
作者
Elisa Domingues,Thomas Brillet,Corinne Vasseur,Virginie Agier,Michael C. Marden,Véronique Baudin‐Creuza
出处
期刊:Plasmid
[Elsevier]
日期:2009-01-01
卷期号:61 (1): 71-77
被引量:6
标识
DOI:10.1016/j.plasmid.2008.09.006
摘要
To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-alpha-[SD]-beta. This vector allows the co-expression of alpha-Hb as a fusion protein with Glutathione S-Transferase (GST-alpha-Hb) and beta-Hb with an additional methionine at the N-terminal extremity (rbeta-Hb). These proteins were solubilized as GST-alpha-Hb/rbeta-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-alpha-Hb/rbeta-Hb complex and recombinant Hb A exhibit the same allosteric behavior as for native Hb A. The GST moiety, which does not modify the function of the complex, can be easily eliminated by cleavage by the PreScission Protease. After cleavage during the rapid purification procedure, over 20mg of recombinant Hb per liter of culture were obtained, more than double the yield of previous co-expression systems. This polycistronic vector system, which offers the additional advantage of a very rapid purification, is especially well suited for the study of abnormal, unstable globins in order to better understand the associated pathology.
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