Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-04-25
2002-09-24
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
Reexamination Certificate
active
06455676
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel hemoglobin mutants having one or more desired functions, including reduced nitric oxide scavenging and increased soluble expression. The invention further relates to methods of increasing soluble expression of recombinant hemoglobin by the addition of excess heme.
BACKGROUND OF THE INVENTION
Hemoglobin (Hb) is the oxygen-carrying component of blood that circulates through the bloodstream inside small enucleate cells known as erythrocytes or red blood cells. It is a protein comprised of four associated polypeptide chains that bear prosthetic groups known as hemes. The structure of hemoglobin is well known and described in Bunn & Forget, eds.,
Hemoglobin: Molecular, Genetic and Clinical Aspects
(W. B. Saunders Co., Philadelphia, Pa.: 1986) and Fermi & Perutz “Hemoglobin and Myoglobin,” in Phillips and Richards,
Atlas of Molecular Structures in Biology
(Clarendon Press: 1981).
Expression of various recombinant hemoglobins containing naturally-occurring and non-naturally occurring globin mutants has been achieved. Such expression methods include individual globin expression as described, for example, in U.S. Pat. No. 5,028,588, and di-alpha globin expression created by joining two alpha globins with a glycine linker through genetic fusion coupled with expression of a single beta globin gene to produce a pseudotetrameric hemoglobin molecule as described in WO 90/13645 and Looker et al.,
Nature
356:258-260 (1992). Other modified recombinant hemoglobins are disclosed in PCT Publication WO 96/40920. Similar to other heterologous proteins expressed in
E. coli,
recombinant hemoglobins have N-terminal methionines, which in some recombinant hemoglobins replace the native N-terminal valines.
The process cost of producing recombinant hemoglobin is affected by the yield of soluble protein. Economic production of heterologous protein in
E.coli
is especially challenging when the protein must not only be soluble and functional, and it is also composed of multiple subunits as for recombinant hemoglobin. In addition, recombinant hemoglobins require the enhanced presence of essential co-factors (prosthetic groups) such as heme and flavins through supplementation or increased endogenous production. In
E.coli,
soluble accumulation of recombinant hemoglobin is limited by heme availability as indicated by the fact that without heme supplementation, addition of &dgr;-ALA, a heme precursor, increases heme and protein accumulation. Consequently, methods of increasing soluble yield are highly desirable. However, prior to the present invention, the effect of mutations on soluble
E. coli
expression had not been studied nor were the determinants of soluble yield thoroughly understood. Thus, a need exists for methods of increasing soluble yield of recombinant hemoglobin.
A need also exists for methods of reducing scavenging of nitric oxide (“NO”) by extracellular hemoglobin. Mild hypertension has sometimes been observed following administration of certain extracellular hemoglobin solutions. It is believed by many that the hypertension is due to depletion of nitric oxide (“NO”) in the wall of the vasculature based in part on the known high affinity of deoxyhemoglobin for NO (Schultz et al.,
J. Lab. Clin. Med.
122:301-308 (1993); Thompson et al.,
J. Appl. Physiol.
77:2348-2354 (1994); Rooney et al.,
Anesthesiology
79:60-72 (1993)). Extravasation of the hemoglobin into endothelial cells or interstitial spaces may cause significant consumption of NO (Gould et al.,
World J. Surg.
20: 1200-1207 (1996)). A recent study also suggests that the oxidative reaction of NO with the bound O
2
of oxyhemoglobin may be of greater significance in vivo than simple binding to the iron atom as reported in Eich et al.,
Biochemistry
35: 6976-6983 (1996). Eich et al. showed that steric hinderance introduced by substitution of amino acids adjacent to bound oxygen can markedly lower the rate of NO-induced oxidation.
Nitric oxide acts as a chemical messenger in the control of many important processes in vivo, including neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. The biological actions of nitric oxide are mediated by binding to and activation of soluble guanylyl cyclase, which initiates a biochemical cascade resulting in a variety of tissue-specific responses (Feldman et al.,
Chem. Eng. News Dec:
26-38 (1993)).
Elucidating the functions of nitric oxide has depended largely on inhibition of the NO-generating enzyme, nitric oxide synthase. Most conclusions about the effects of cell-free hemoglobin have been drawn based on experiments involving NO synthase inhibitors and/or NO donors.
While the rapid, high-affinity binding of nitric oxide to deoxyhemoglobin is well known, the importance of the oxidative reaction between NO and oxyhemoglobin is not as widely appreciated. In this reaction, the NO molecule does not bind to the heme, but reacts directly with the bound oxygen of the HbO
2
complex to form methemoglobin and nitrate (Doyle et al.,
J. Inorg. Biochem.
14: 351-358 (1981)). The chemistry is analogous to the rapid reaction of NO with free superoxide in solution (Huie et al.,
Free Rad. Res. Comms.
18: 195-199 (1993)). Both the heme iron and nitric oxide become oxidized by the bound oxygen atoms, and the reaction occurs so rapidly that no replacement of O
2
by NO is observed (Eich et al., supra.).
Since nitric oxide is produced and consumed on a continuous basis, there is a natural turnover of NO in vivo. When a cell-free hemoglobin is administered, the balance between NO production and consumption is altered by reactions with hemoglobin. The most relevant parameter for NO scavenging by oxyhemoglobin is the rate of reaction with NO, not the position of the Hb allosteric (R/T) equilibrium. The oxidative reaction is irreversible, and NO binding to deoxyhemoglobin is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours (Moore et al.,
J. Biol. Chem.
251: 2788-2794 (1976).
Once an NO molecule reacts with oxyhemoglobin or deoxyhemoglobin, it is eliminated from the pool of signal molecules causing certain adverse conditions. For example, hemoglobin can bind nitric oxide causing the prevention of vascular relaxation and potentially leading to hypertension that is sometimes observed after administration of certain extracellular hemoglobin solutions. In addition, the ability of NO to oxidize oxyhemoglobin producing peroxynitrite and methemoglobin could also lower free concentrations of NO and lead to hypertension.
Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation. Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A
2
and serotinin. These compounds may act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in an significant vasoconstriction.
In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn can lead to cell wall damage. Endothelial cell wall damage has been observed with the infusion of certain hemoglobin solutions (White et al.,
J. Lab. Clin. Med.
108:121-181 (1986)).
Accordingly, a need exists for new hemoglobin mutants with decreased NO-scavenging while still functioning as an effective oxygen carrying agent. The present invention satisfies this need as well as the need for increased soluble yield of recombinant hemoglobin.
SUMMARY OF THE INVENTION
The invention relates to hemoglobin mutations that confer certain desired functions, such as increased yield of soluble Hb and reduced NO scavenging. The hemoglobin mutants can be designed to include these functions separately or in combination.
Therefore, in one aspect, the invention is directed to intro
Doherty Daniel H.
Glascock Christopher B.
Lemon Douglas D.
Mathews Antony J.
Olson John S.
Baxter Biotech Technology SARL
Carlson Karen Cochrane
Senniger Powers Leavitt & Roedel
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