Hemoglobin Rainier is a naturally occurring
hemoglobin variant in which the beta 145
tyrosine is substituted with
cysteine. The alpha and beta Rainier
globin cDNAs were cloned in a high copy number vector and expressed in Saccharomyces cerevisiae under the control of
galactose-regulated hybrid promoters. Using this system, we have expressed individual alpha and beta Rainier
globin chains. Coexpression of both alpha and beta Rainier cDNAs resulted in the production of a functional
hemoglobin molecule. Purification of the
recombinant protein was accomplished by ion exchange chromatography. The N-termini of the alpha and beta chains were correctly processed, and the molecular mass, as determined 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
hemoglobin A0. The purified recombinant
hemoglobin molecule was shown to have an elevated
oxygen affinity and a reduced cooperativity as previously reported for natural
hemoglobin Rainier. Production of recombinant
hemoglobin and especially
hemoglobin variants like
hemoglobin Rainier has the potential to facilitate use of
hemoglobin as a
blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of
hypotension associated with
septic shock.