Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin
To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-α-[SD]-β. This vector allows the co-expression of α-Hb as a fusion protein with Glutathione S-Transferase (GST-α-Hb) and β-Hb with an additional met...
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Published in | Plasmid Vol. 61; no. 1; pp. 71 - 77 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
2009
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Subjects | |
Online Access | Get full text |
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Summary: | To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-α-[SD]-β. This vector allows the co-expression of α-Hb as a fusion protein with Glutathione
S-Transferase (GST-α-Hb) and β-Hb with an additional methionine at the N-terminal extremity (rβ-Hb). These proteins were solubilized as GST-α-Hb/rβ-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-α-Hb/rβ-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 20
mg 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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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0147-619X 1095-9890 |
DOI: | 10.1016/j.plasmid.2008.09.006 |