DNA encoding fused di-alpha globins and use thereof

Details DNA encoding fused di-alpha globins and use thereof DNA encoding fused di-alpha globins and use thereof

1995-05-30

2004-12-07

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S320100, C536S023100, C530S385000

Reexamination Certificate

active

06828125

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the intracellular assembly of a hemoglobin-like protein in biologically functional, substantially soluble form through co-expression of alpha and beta globin-like polypeptides in bacterial or yeast cells.
It further relates to the genetic cross-linking of the two alpha subunits of hemoglobin to form a novel polypeptide, di-alpha globin, which may be considered a partially assembled intermediate leading to a hemoglobin-like protein, and the use of this compound in the production of synthetic hemoglobins having an increased intravascular half-life as compared to stroma-free hemoglobins. It also relates to the analogous polypeptide di-beta globin.
INFORMATION DISCLOSURE STATEMENT
It is not always practical to transfuse a patient with donated blood. In these situations, use of a red blood cell substitute is desirable. The product must effectively transport O
2
, just as do red blood cells. (“Plasma expanders”, such as dextran and albumin, do not transport oxygen.) The two types of substitutes that have been studied most extensively are hemoglobin solutions and fluorocarbon emulsions.
A. Structure and Function of Hemoglobin
Hemoglobin (Hgb) is the oxygen-carrying component of blood. Hemoglobin circulates through the blood stream inside small enucleate cells called erythrocytes (red blood cells). Hemoglobin is a protein constructed from four associated polypeptide chains, bearing prosthetic groups known as hemes. The erythrocyte helps maintain hemoglobin in its reduced, functional form. The heme iron atom is susceptible to oxidation, but may be reduced again by one of two enzyme systems within the erythrocyte, the cytochrome b
5
and glutathione reduction systems.
About 92% of the normal adult human hemolysate is hemoglobin A (designated alpha2 beta2, because it comprises two alpha and two beta chains). The alpha chain consists of 141 amino acids. The iron atom of the heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole of His 87 (the “proximal histidine”). The beta chain is 146 residues long and heme is bound to it at His 92. Apohemoglobin is the heme-free analogue of hemoglobin; it exists predominantly as the &agr;&bgr;-globin dimer.
Separated, heme-free, alpha and beta globins have been prepared from the heme-containing alpha and beta subunits of hemoglobin. The separated heme-free globin chains are folded very differently, even though, the heme-containing subunits are highly similar in secondary structure and basic folding features. This shows that the binding of the prosthetic heme group to globin subunits has quite different effects on alpha and beta globin. Yip, et al., J. Biol. Chem., 247: 7237-44 (1972).
Native human hemoglobin has been fully reconstituted from separated heme-free alpha globin and beta globin and hemin. Preferably, heme is first added to the alpha globin subunit. The heme-bound alpha globin is then complexed to the heme-free beta subunit. Finally, heme is added to the half-filled globin dimer, and tetrameric hemoglobin is obtained. Yip, et al., PNAS (USA), 74: 64-68 (1977).
In cell-free systems prepared from unfractionated rabbit reticulocyte hemolysates, globin is actively synthesized for approximately five minutes, and then protein synthesis abruptly ceases. Prior addition of hemin prevents or delays the cessation of synthetic activity, as a result of the effect of hemin on an inhibitory protein known as “hemin-regulated inhibitor” (HRI). Hemin deficiency has a more severe effect on alpha chain synthesis than on a beta chain synthesis as alpha globin mRNA is less efficient than beta globin mRNA in initiating polypeptide chain synthesis. It has been speculated that alpha chains are released from their site of synthesis only in the presence of free beta chains, which immediately complex the released alpha chains to form &agr;&bgr; globin dimers. These then combine with heme to form tetrameric hemoglobin. Winterhalter and Huehns, J. Biol. Chem., 239: 3699 (1964). It is certainly known that the addition of heme to &agr;&bgr; globin dimers (apohemoglobin) leads to the rapid formation of hemoglobin.
The human alpha and beta globin genes reside on chromosomes 16 and 11, respectively. Bunn and Forget,
Hemoglobin: Molecular, Genetic and Clinical Aspects
(W. B. Saunders Co., Philadelphia Pa. 1986). Both genes have been cloned and sequenced. Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha globin genomic DNA); Marotta, et al., J. Biol. Chem.; 252: 5040-53 (1977) (beta globin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomic DNA).
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule (two alpha globins and two beta globins in the case of hemoglobin A), and this cooperativity greatly facilitates efficient oxygen transport. Cooperativity, achieved by the so-called heme-heme interaction, allows hemoglobin to vary its affinity for oxygen. Hemoglobin reversibly binds up to four moles of oxygen per mole of hemoglobin.
Oxygen-carrying compounds are frequently compared by means of a device known as an oxygen dissociation curve. This curve is obtained when, for a given oxygen carrier, oxygen saturation or content is graphed against the partial pressure of oxygen. For hemoglobin, the percentage of saturation increases with partial pressure according to a sigmoid relationship. The P
50
is the partial pressure at which the oxygen-carrying solution is half saturated with oxygen. It is thus a measure of oxygen-binding affinity; the higher the P
50
, the more loosely the oxygen is held.
When the oxygen dissociation curve of an oxygen-carrying solution is such that the P
50
is less than that for whole blood, it is said to be “left-shifted.”
The oxygen affinity of hemoglobin is lowered by the presence of 2,3-diphosphoglycerate (2,3-DPG), chloride ions and hydrogen ions. Respiring tissue releases carbon dioxide into the blood and lowers its pH (i.e. increases the hydrogen ion concentration), thereby causing oxygen to dissociate from hemoglobin and allowing it to diffuse into individual cells.
The ability of hemoglobin to alter its oxygen affinity, increasing the efficiency of oxygen transport around the body, is dependent on the presence of the metabolite 2,3-DPG. Inside the erythrocyte 2,3-DPG is present at a concentration nearly as great as that of hemoglobin itself. In the absence of 2,3-DPG “conventional” hemoglobin binds oxygen very tightly and would release little oxygen to respiring tissue.
Aging erythrocytes release small amounts of free hemoglobin into the blood plasma where it is rapidly bound by the scavenging protein haptoglobin. The hemoglobin-haptoglobin complex is removed from the blood and degraded by the spleen and liver.
B. Blood Substitutes, Generally
It is clear from the above considerations that free native hemoglobin A, injected directly into the bloodstream, would not support efficient oxygen transport about the body. The essential allosteric regulator 2,3-DPG is not present in sufficient concentration in the plasma to allow hemoglobin to release much oxygen at venous oxygen tension.
Nonetheless, solutions of conventional hemoglobin have been used as red blood cell substitutes. The classic method of preparing hemoglobin solutions employs outdated blood. The red cells are lysed and cellular debris is removed, leaving what is hopefully “stroma-free hemoglobin” (SFH).
Several basic problems have been observed with this approach. The solution must be freed of any toxic components of the red cell membrane without resorting to cumbersome and tedious procedures which would discourage large-scale production. DeVenuto, “Appraisal of Hemoglobin Solution as a Blood Substitute”,
Surgery, Gynecology and Obstetrics
, 149: 417-436 (1979).
Second, as expected such solutions are “left-shifted” (lower P
50
) as compared to whole blood. Gould, et al., “The Development of Polymerized Pyridoxylated Hemoglobin Solution as a Red Cell Substitute”,
Ann. Emerg. Med
. 15: 1416-1419 (Dec. 3, 1986). As a result, the oxygen affinity is too high to unload

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