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

Abstract

The α-subunits of human hemoglobin (Hb) have been more difficult to express than β-chains owing to the high instability of α-chains. Here, we describe the production in Escherichia coli of a soluble recombinant α-Hb with human α-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-α-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express α-hemoglobin and human AHSP. While the GST-α-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-α-AHSP permits the co-expression of soluble GST-α-Hb and GST-AHSP. The α-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 α-Hb complex were similar to those of recombinant AHSP/native α-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. © The Author 2006. Published by Oxford University Press. All rights reserved.