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PEDS Advance Access published online on January 3, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzj006
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received October 20, 2005
Revised December 6, 2005
Accepted December 6, 2005

Article

High-yield expression in Escherichia coli of soluble human {alpha}-hemoglobin complexed with its molecular chaperone

Corinne Vasseur-Godbillon 1, Djemel Hamdane 1, Michael C. Marden 1, and Véronique Baudin-Creuza 1 *

1 INSERM U473, 78 rue du Général Leclerc, F-94275 Le Kremlin-Bicêtre Cedex, France

* To whom correspondence should be addressed.
Véronique Baudin-Creuza, E-mail: baudin{at}kb.inserm.fr


  Abstract

The {alpha}-subunits of human hemoglobin (Hb) have been more difficult to express than {beta}-chains owing to the high instability of {alpha}-chains. Here, we describe the production in Escherichia coli of a soluble recombinant {alpha}-Hb with human {alpha}-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-{alpha}-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express {alpha}-hemoglobin and human AHSP. While the GST-{alpha}-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-{alpha}-AHSP permits the co-expression of soluble GST-{alpha}-Hb and GST-AHSP. The {alpha}-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant {alpha}-Hb complex were similar to those of recombinant AHSP/native {alpha}-Hb complex as assessed by UV/visible and CO or O2 binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.

Keywords: {alpha}-hemoglobin-stabilizing protein (AHSP); Escherichia coli AHSP and {alpha}-hemoglobin expression system; glutathione S-transferase protein; human {alpha}-hemoglobin; human hemoglobin; molecular chaperone AHSP.
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