High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone.
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 O(2) 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.
Vasseur-Godbillon
Corinne
C
INSERM U473, Le Kremlin-Bicêtre, France.
Hamdane
Djemel
D
Marden
Michael C
MC
Baudin-Creuza
Véronique
V
D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
C468768:AHSP protein, human; D001798:Blood Proteins; D008024:Ligands; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase