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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin.
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Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin.

机译：一种新型多顺反子载体的构建，用于人类血红蛋白的过表达和快速纯化。

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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.
机译：为了促进对人类血红蛋白（Hb A）的结构-功能关系的研究，我们开发了一种新的血红蛋白表达载体pGEX6P-alpha- [SD] -beta。该载体允许α-Hb作为融合蛋白与谷胱甘肽S-转移酶（GST-α-Hb）和β-Hb与另外的蛋氨酸在N端末端（rbeta-Hb）共表达。将这些蛋白溶解为GST-α-Hb/ rbeta-Hb复合物形式，并通过固定在谷胱甘肽上的亲和层析一步纯化。 CO结合动力学研究表明，GST-alpha-Hb / rbeta-Hb复合物和重组Hb A与天然Hb A表现出相同的变构行为。不改变复合物功能的GST部分可以很容易地通过PreScission蛋白酶切割消除。在快速纯化过程中裂解后，每升培养物可获得超过20mg的重组Hb，是先前共表达系统产量的两倍以上。这种多顺反子载体系统具有非常快速纯化的额外优势，特别适合研究异常，不稳定的球蛋白，以便更好地了解相关的病理学。

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