Production, purification, and characterization of recombinant human hemoglobin rainier expressed in Saccharomyces cerevisiae.
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.
Motwani
N
N
Apex Bioscience, Inc., Research Triangle Park, North Carolina 27709-2847, USA.
Talarico
T
T
Jain
S
S
Bajwa
W
W
Blackburn
R
R
Nwosu
V
V
Holland
M
M
DeAngelo
J
J
Privalle
C
C
Keng
T
T
D002874:Chromosome Mapping; D005822:Genetic Vectors; D006455:Hemoglobins, Abnormal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010449:Peptide Mapping; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae
D006455:Hemoglobins, Abnormal; D011994:Recombinant Proteins; C046444:hemoglobin Rainier