Open AccessStructural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae
Author(s) -
COGHLAN David,
JONES Gareth,
DENTON Katharine A.,
WILSON Michael T.,
CHAN Bernard,
HARRIS Roy,
WOODROW John R.,
OGDEN Jill E.
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17126.x
Subject(s) - recombinant dna , tetramer , saccharomyces cerevisiae , biochemistry , bohr effect , chemistry , yeast , hemoglobin , enzyme , gene , oxygen–haemoglobin dissociation curve
Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae , has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier.