PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN HEMOGLOBIN RAINIER EXPRESSED IN SACCHAROMYCES-CEREVISIAE

Hemoglobin Rainier is a naturally occurring hemoglobin variant in whic h the beta 145 tyrosine is substituted with cysteine. The alpha and be ta(Rainier) globin cDNAs were cloned in a high copy number vector and expressed in Saccharomyces cerevisiae under the control of galactose-r egulated hybrid promoters. Using this system, we have expressed indivi dual alpha and beta(Rainier) globin chains. Coexpression of both alpha and beta(Rainier) cDNAs resulted in the production of a functional he moglobin molecule. Purification of the recombinant protein was accompl ished by ion exchange chromatography. The N-termini of the alpha and b eta chains were correctly processed, and the molecular mass, as determ ined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived he moglobin A(0). The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as pre viously reported for natural hemoglobin Rainier. Production of recombi nant hemoglobin and especially hemoglobin variants like hemoglobin Rai nier has the potential to facilitate use of hemoglobin as a blood subs titute as well as in specific applications, such as for use as a thera peutic agent in the treatment of hypotension associated with septic sh ock. (C) 1996 Academic Press, Inc.