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...
Patent
1998-04-22
2000-09-05
Carlson, Karen Cochrane
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...
435 691, C07K 14805, C12P 2106
Patent
active
06114505&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to mutant recombinant hemoglobins containing mutations which reduce the rate of loss of heme from the globin moiety. This invention further relates to improved yields of hemoglobin by expression of certain mutant subunits.
BACKGROUND OF THE INVENTION
The current blood banking system has inherent risks and serious limitations. Blood typing errors, transmission of bacterial agents, and viral infections such as HIV-1 and hepatitis A, B, and AB pose life threatening dangers to transfusion patients. In addition, availability of donors, requirement for specific blood types, short shelf life of red blood cells, and need for refrigeration all limit the accessibility of a transfusion to a patient. Development of a stable blood substitute could eliminate the risks of the current blood banking system and increase the availability of transfusions to patients in most environments.
In addition, an oxygen carrying blood substitute can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, and can also support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution can be used in most situations where red blood cells or plasma expanders are currently utilized. An oxygen-transporting hemoglobin-based solution could also be used to temporarily augment oxygen delivery during or after pre-donation of autologous blood prior to the return of the autologous blood to the patient.
However, several obstacles must be overcome in the development of an optimal hemoglobin-based oxygen carrier, including: (1) inhibition of tetramer to dimer dissociation; (2) reduction of hemoglobin oxygen affinity; (3) inhibition of autooxidation; (4) inhibition of heme loss; and (5) increased stability of the apoglobin tertiary structures.
Thus far, most hemoglobin-based blood substitute designs have successfully focused on preventing tetramer dissociation and reducing oxygen affinity through chemical and genetic techniques (Winslow, R. M. (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). Gawryl and coworkers (Gawryl, M., Clark, T., and Rausch, C., in Red Cell Substitutes: The Proceedings of the Second International Symposium of Red Blood Cell Substitutes (S. Sekiguchi, ed.) pp. 28-40, Tokyo:Kindai Shupann, 1991) chemically cross-linked bovine hemoglobin using glutaraldehyde to prevent tetramer dissociation. Besides being very abundant, bovine hemoglobin was chosen because its oxygen affinity is regulated by chloride ions, and, as a result, has a relatively high P.sub.50 value, which is the same inside or outside a red blood cell. Chatterjee et al. (Chatterjee, R., Welty, E. V., Walder, R. Y., Pruitt, S. L., Rogers, P. H., Arnone, A., and Walder, J. A. (1986) J. Biol. Chem. 261, 9929-9937) chemically crosslinked human hemoglobin using (3,5-dibromosalicyl)fumarate under conditions which also caused a two fold decrease in the oxygen affinity of the modified protein. A genetic approach is discussed by Hoffman et al., who used recombinant hemoglobin genes and an E. coli. expression system (Hoffman et al., WO 90/13645). They genetically linked the C-terminal residue of one alpha subunit to the N-terminus of the other alpha subunit using a flexible glycine residue, producing a single alpha.sub.1 -alpha.sub.2 subunit (Looker, D., et al. Methods in Enzymology 231, 364-374, 1994). The tandem alpha globin gene was then combined with a copy of the beta globin gene and placed under the control of a single promoter to form a hemoglobin operon. To decrease the oxygen affinity, they also incorporated the Presbyterian mutation into the beta subunits. Presbyterian mutation refers to the beta(G10)Asp.fwdarw.Lys substitution which causes a reduction in oxygen affinity in both hemoglobin subunits. The final protein was designated rHb1.1 and has a P.sub.50 value similar to that observed for intact red blood cells.
Inhibition of tetramer dis
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Hargrove Mark S.
Olson John S.
Whitaker Timothy L.
Carlson Karen Cochrane
William Marsh Rice University
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