Recombinant human sickle hemoglobin expressed in yeast.

Abstract

Sickle hemoglobin has been expressed in the yeast Saceharomyces cerevwisa after site-diected mutagenesis of a plasmid conti normal human a-and I3-globin genes.Cassette mutagenesis of this plasnid was achieved by inserting a DNA fragment containing the 3-globin gene in the replicative form of M13mpl8 to make a point mutation and then recondfIy the original paid containing the mutated -globin gene. Pure recombinant hemolbin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel dectrophoresi, iboectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and finctional properties (Pso, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranlational processing yields the same N-terminal valine residues of hemoglobin a-and a-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 hunctions, 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 as ets of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering ofthis system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.The genetic mutation underlying sickle cell anemia leads to the replacement of Glu-6 on the (-chain with a valine residue (1, 2). The substitution of a hydrophobic side chain for a hydrophilic one on the exterior ofthe protein (3) promotes the aggregation of deoxygenated hemoglobin (Hb) tetramers (4, 5) that eventually distort the erythrocyte into a sickled form in the venous circulation (6). One approach toward treatment of sickle cell anemia focuses on the HbS molecule itself by reacting it with chemical modifiers that directly alter the functional properties of the protein and indirectly reduce aggregation in vitro (7,8). The number and nature of such susceptible sites has been limited to those hydrophilic side chains with enhanced reactivity because of their location in the protein (8, 9). The availability of recombinant DNA technology now permits studies at any site on the HbS tetramer, such as hydrophobic amino acid side chains heretofore not possible to modify by other methods. For sickle cell HbS, the ability to alter hydrophobic sites is especially important since the initial and some of the subsequent stages of aggregation involve hydrophobic interactions. Although the identity of some of these contact sites in the aggregate is known (3)(4)(5)10), there is a lack of information on other contact sites and their contribution to the overall strength of the ...

Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala

“…In this system, Sacchoromyces cerevisiae carrying a plasmid containing the human a-and 0-globin cDNA sequences permits correct N-terminal processing and a soluble H b tetramer is produced (Ogden et al, 1994). The oxygen equilibrium curves of recombinant and natural sickle H b a t high protein concentration were superimposable, and there was a high degree of cooperativity as indicated by Hill coefficients of 3.3 (Martin de Llano et al, 1993a). Furthermore, the gelation properties of recombinant and natural sickle H b were also the same at the high H b concentrations that occur in the red cell.…”

“…The recombinant Hb was expressed in yeast, which was grown and collected as described previously (Martin de Llano et al, 1993b). The growth of the yeast was not adversely affected by the presence of the mutant Hb.…”

“…The Hb E6V/L88A(/3) double mutant prepared as described above, the recombinant HbS obtained as described previously (Martin de Llano et al, 1993a, 1993b, as well as natural HbA and natural HbS from human red cells were each purified by HPLC and subjected to isoelectric focusing using the pH 6-8 Hb-Resolve system (Isolab). This system is sufficiently discriminating to separate various types of Hb and to detect any impurities, if present.…”

“…The growth of the yeast was not adversely affected by the presence of the mutant Hb. Purification was achieved on the CM-52 and Synchropak CM-300 HPLC columns as described in Martin de Llano et al (1993a).…”

“…Hence, Leu-88 was changed to an Ala residue because such a substitution would not alter the hydrophobic character of this important primary binding site for aggregation. This mutation was achieved with the yeast expression system recently described for sickle H b (Wagenbach et al, 1991;Martin de Llano et al, 1993a, 1993b. In this system, Sacchoromyces cerevisiae carrying a plasmid containing the human a-and 0-globin cDNA sequences permits correct N-terminal processing and a soluble H b tetramer is produced (Ogden et al, 1994).…”