Recombinant human sickle hemoglobin expressed in yeast.
Sickle hemoglobin has been expressed in the yeast Saccharomyces cerevisiae after site-directed mutagenesis of a plasmid containing normal human alpha- and beta-globin genes. Cassette mutagenesis of this plasmid was achieved by inserting a DNA fragment containing the beta-globin gene in the replicative form of M13mp18 to make a point mutation and then reconstituting the original plasmid containing the mutated beta-globin gene. Pure recombinant hemoglobin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel electrophoresis, isoelectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and functional properties (P50, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranslational processing yields the same N-terminal valine residues of hemoglobin alpha- and beta-chains, but in bacterial expression systems the N terminus is extended by an additional amino acid because the initiator methionine residue is retained. Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological functions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies. Hence, mutagenesis employing this expression system should permit definitive assignments of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering of this system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.
Martin de Llano
J J
JJ
Rockefeller University, New York, NY 10021-6399.
Schneewind
O
O
Stetler
G
G
Manning
J M
JM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006451:Hemoglobin, Sickle / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry
D000596:Amino Acids; D006451:Hemoglobin, Sickle; D011994:Recombinant Proteins; D010100:Oxygen