Functional properties of human hemoglobins synthesized from recombinant mutant .beta.-globins

Doyle, Michael L.; Lew, George; Young, Alice De; Kwiatkowski, Laura D.; Wierzba, Anita; Noble, Robert W.; Ackers, Gary K.

doi:10.1021/bi00151a033

Cited by 53 publications

(77 citation statements)

References 39 publications

(60 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The synthetic human a-and 3-globin genes for normal Hb (17) and for a few other mutant Hbs (18)(19)(20)(21)(22)27) have been expressed in E. coli. However, the Hb produced in this system is not processed at its N terminus in the same way that human Hb is processed in human reticulocytes.…”

Section: Discussionmentioning

confidence: 99%

Recombinant human sickle hemoglobin expressed in yeast.

Llano

Schneewind

Stetler

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

confidence: 99%

Recombinant human sickle hemoglobin expressed in yeast.

Llano

Schneewind

Stetler

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…46 The Di␣Hb and CpHb were expressed in E coli DH5␣ cells. The cultures were grown in 1 L of 2XYT media (16 g tryptone, 10 g yeast extract, and 5 g NaCl/L) containing 200 g/mL ampicillin and 0.5 mM …”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Intersubunit circular permutation of human hemoglobin

Sanders

Sligar

2002

Blood

View full text Add to dashboard Cite

For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species. The use of total gene synthesis has resulted in very high levels of functional human Hb expression in Escherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight. In this communication, we report a novel method for linking alpha chains. By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity. The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic. IntroductionA mechanistic and predictive understanding of how a primary sequence can fold to a single tertiary structure remains undefined. Since a thorough understanding of this process will enable the rational design of unique protein structures, this remains an area of active research. One theory suggests that short continuous regions within a protein develop local interactions early in the folding process to minimize the number of accessible structures and catalyze the formation of a functional protein. Radical perturbations of protein structure by circular permutation is one technique being used in an attempt to explore this issue. In addition, utilization of circular permutation itself can be exploited in the design of new protein structures.A circularly permuted protein is created in 2 steps. First, the original termini are linked to form a circular polypeptide. New termini are then created by cleavage of a peptide bond at a location distant from the original termini. Goldenberg and Creighton engineered the first circularly permuted sequence by chemically condensing the termini of bovine pancreatic trypsin inhibitor (BPTI) and generating new termini by limited proteolysis. 1 This circularly permuted variant was shown to refold, in vitro, to a native functional conformation. This seminal experiment suggested that the location of the termini has little effect on the final 3-dimensional structure of the protein. Luger and coworkers genetically circularly permuted phosphoribosyl anthranilate isomerase. 2 The Escherichia coli translated variant was structurally and functionally similar to the wild-type protein. Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic cha...

show abstract

“…The resulting ␤-globin is insoluble, does not incorporate heme, and has a methionine in place of the normal NH 2 -terminal valine. However, when combined with native ␣-subunits and hemin, the purified ␤-globin chains are incorporated into tetrameric Hb that is functionally and structurally very similar to native HbA (12,13,15). Specific isotopic enrichment of valine backbone nitrogen atoms was achieved by supplementing the LB with 60 mg͞liter 15 N-labeled valine (Cambridge Isotope Laboratories, Cambridge, MA).…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, triple resonance experiments with 2 H decoupling can yield backbone assignments for uniformly 13 C, 15 Nlabeled and highly deuterated proteins up to 60 kDa (8). Recent implementation of transverse relaxation-optimized spectroscopy (TROSY) in triple resonance experiments has improved sensitivity severalfold for N-labeled proteins (9)(10)(11), and complete backbone assignments of proteins up to 110 kDa has been shown to be feasible (9,10).…”

mentioning

confidence: 99%

“…We have begun by using the expression system of Hernan et al (12) [which yields functionally and structurally faithful ␤(V1M)-chains] to 15 N-label the backbone amide nitrogens of the ␤-chain Val residues (3,13). Valine is an ideal residue for such a site-specific labeling experiment because it is one of the most abundant amino acid residues in both the ␣-and ␤-chains of Hb (Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation