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PEDS Advance Access originally published online on January 3, 2006
Protein Engineering Design and Selection 2006 19(3):91-97; 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

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

Corinne Vasseur-Godbillon, Djemel Hamdane, Michael C. Marden and Véronique Baudin-Creuza1

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

1 To whom correspondence should be addressed. E-mail: baudin{at}kb.inserm.fr

The {alpha}-subunits of human hemoglobin (Hb) have been more difficult to express than ß-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

Received October 20, 2005; revised December 6, 2005; accepted December 6, 2005.

Edited by Hans Thogerson


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