Described are preferred oxygen-binding heme proteins which include at least one hemoglobin molecule incorporating at least one circularly permuted globin, especially an alpha globin. More preferred heme proteins of the invention include high molecular weight hemoglobin multimers. Also described are polynucleotides encoding proteins of the invention, and vectors and host cells including the same.

BACKGROUND OF THE INVENTION
 The present invention relates generally to oxygen-binding heme proteins,
 and in particular to such proteins incorporating one or more hemoglobin
 tetramers incorporating at least one functional, circularly-permuted
 globin.
 As further background, blood transfusions allow trauma patients means to
 replenish blood loss, surgery patients to enter longer procedures with
 less risk, and rescue workers to bring a blood supply to accident victims.
 Although a transfusable blood supply provides many benefits, available
 blood is limited by human donations. In addition, the limited shelf-life
 of whole blood, disease transfer, and mismatched blood typing are problems
 yet to be fully addressed.
 For example, the occurrence of an HIV-contaminated blood supply in the
 1980's heightened awareness for a need to circumvent the problems
 associated with a donated blood supply. Even today, the United States
 Department of Health and Human Services has created a blood safety panel
 to examine many issues relative to donated blood, including HIV and
 hepatitis.
 Transfused blood, containing plasma, white blood cells (leukocytes),
 platelets and red blood cells (erythrocytes), is generally used to carry
 oxygen from the lungs to the rest of the body's cells. A number of oxygen
 carrying solutions are being studied as alternatives to blood
 transfusions. In this regard, an effective blood substitute must satisfy
 three basic requirements. First, it must transport oxygen from lungs to
 tissues. Second, it must remain functional in vivo long enough to be
 effective; and third, it must not elicit harmful side effects. Blood
 substitutes studied to date include perfluorocarbons (Kaufman, R. J.
 (1991) in Biotechnology of Blood (J. Goldstein, e., Ed.) pp. 127-162,
 Butterworth-Heinemann, Boston), chemically modified hemoglobin from
 outdated human blood (Winslow, R. M. (1992) Hemoglobin-based red cell
 substitutes, Johns Hopkins University Press, Baltimore), and recombinant
 hemoglobins produced in microbial and mammalian hosts (Shen, T. -J., Ho,
 N. T., Simplaceanu, V., Zoiu, M., Green, B. N., Tam, M. F., & Ho, C.
 (1963), PNAS USA 90, 8108-8112; Rao, M. J., Schneider, K., Chait, B. T.,
 Chao, T. L., Keller, H. Anderson, S., Manjula, B. N., Kumar, R., &
 Acharya, A. S. (1994) ACBSIB 22, 695-700).
 On the subject of hemoglobin, each hemoglobin molecule is a tetramer of
 four smaller polypeptide subunits known as globins. A heme group, which is
 an iron-protoporphyrin complex, is associated with each polypeptide
 subunit, and is responsible for the reversible binding of a single
 molecule of oxygen. Normal adult hemoglobin is made up of two different
 kinds of polypeptide globins. A first globin, known as alpha globin,
 contains 141 amino acid residues. The second, known as beta globin,
 contains 146 amino acid residues. In normal adult hemoglobin, two of each
 kind of globin are arranged in the form of a truncated tetrahedron which
 has the overall shape of an ellipsoid.
 The overall hemoglobin molecule is a 64,400 kDa protein. X-ray crystal
 structures show the size of HbAo to be about 64 .ANG..times.55
 .ANG..times.50 .ANG. (Fermi, G., Perutz, M. F., Shaanan, B. and Fourme, B.
 (1984) Journal of Molecular Biology 175, 159). The heme prosthetic group
 of each alpha subunit is non-covalently bound to the subunits by Lys E10,
 His CD3, Val E11, and Phe CD1. In beta chains, His CD3 is replaced by Ser
 CD3 The heme contains an Fe++ bound by the proximal histidine. A distal
 histidine hovers over the iron but does not coordinate; however, this
 histidine could sterically and/or electronically hinder the binding of CO,
 which has a higher affinity for heme than O.sub.2, as well as hydrogen
 bond to iron in the deoxystate. The irons in the hemes can oxidize to the
 Fe+++ state, creating a nonfunctional hemoglobin (Bunn, H. F. a. F., B. G.
 (1986) in Hemoglobin-Molecular, Genetic, and Clinical Aspects (Dyson, J.,
 Ed.) pp. 13-19, W. B. Saunders Company, Philadelphia).
 Ligands that bind hemoglobin include CO, NO, CN--, and the most
 physiologically relevant ligand, O.sub.2. Oxygen binding occurs in a
 sygmoidal pattern, demonstrating the cooperativity of multiple ligand
 binding. It has been shown that hemoglobin can exist in at least two
 states, T and R. The T state is associated with the deoxygenated state of
 hemoglobin, while the R state is associated with ligand bound hemoglobin.
 A number of models have been offered to describe the shift from T to R
 when ligand is bound. Two primary models describe the change in states as
 either a concerted change from T to R or a sequential change of subunits
 from T to R as ligand is bound. The concerted model proposed by Monod,
 Wynman, and Changeux describes cooperativity resulting from the entire
 tetramer converting from T to R (Monod, J., Wyman, J., and Changeux, J.
 -P. (1965) Journal of Molecular Biology 12, 88-118). The induced fit mode
 describes cooperativity as the result of an R state, ligand bound subunit
 inducing a neighboring T state subunit to alter to the R conformation
 (Koshland, D. E., Nemethy, G. and Filmer, D. (1966) Biochemistry 5,
 365-385). Recently, Ackers and co-workers have proposed a symmetry model
 for T to R transition which provides evidence for an intermediate state in
 T to R transition (Ackers, G. K., Doyle, M. L., Myers, D., and Daugherty,
 M. A. (1992) Science 255, 54-63). The eight intermediate ligation states
 have been studied using metal-substituted hemes that are unable to bind
 ligand. The evidence demonstrates the steepest free energy change occurs
 when a subsequent ligand binds the alternate alpha/beta dimer.
 Ligand affinity is also dependent on a number of allosteric effectors. The
 effectors that lower oxygen affinity include protons (Bohr effect), 2,3
 diphosphoglycerate, and chloride ions. The physiological relevance of the
 effectors is to enhance oxygen delivery to metabolically active cells that
 produce CO.sub.2.
 Modification of human hemoglobin has been widely investigated as a means to
 provide a blood substitute and for other uses. Hemoglobin is a
 well-characterized protein, and can be altered to meet the basic
 requirements for an effective and safe blood substitute. Chemically
 modified, and more recently, recombinant forms of hemoglobin, are
 currently being tested in various stages of clinical trials.
 Some problems arise from overproduction of recombinant hemoglobin in
 prokaryotes and eukaryotes. In humans, methionine aminopeptidase
 recognizes small, hydrogphobic residues as a signal to cleave (Hernan, R.
 A., Hui, H. L., Andracki, M. E., Noble, R. W., Sligar, S. G., Walder, J.
 A., & Walder, R. Y. (1992), Biochemistry 31, 8619-8628). Therefore, the
 first amino acid in postranslationally modified human hemoglobin is a
 valine. However, during the expression of human hemoglobin in E. coli, the
 initial methionine is not cleaved. Further, E. coli methionine peptidase
 recognizes small polar side chains, and expression in E. coli essentially
 adds a methionine to the primary sequence of both alpha and beta chains.
 This issue has been dealt with in two ways. A yeast expression system has
 been utilized in which the initial methionine is cleaved (Wagenbach, M.,
 O'Roueke, K., Vitez, L., Wieczorek, A., Hoffman, S., Durfee, S., Tedesco,
 J., & Stetler, G. (1991) Bio-Technology 9, 57-61). In prokaryotic
 production, the replacement of the first amino acid - valine - with a
 methionine was used in both alpha and beta chains (recombinant hemoglobin
 des-val) to produce a protein functionally similar to HbAo (Hernan, R. A.,
 Hui, H. L., Andracki, M. E., Noble, R. W., Sligar, S. G., Walder, J. A., &
 Walder, R. Y. (1992), Biochemistry 31, 8619-8628).
 It has been reported that these overproduced hemoglobins are misassembled
 in the yeast and E. coli (Hernan, R. A., & Sligar, S. G. (1995) JBC, 270,
 26257-26264). The misassembled tetramer initially binds ligand similarly
 to wild type hemoglobin, but over time drifts to different tetramer
 substrates that bind ligand at different rates. The drift appears to be
 time and temperature dependent, and protein stored at -70.degree. C. still
 encounters a drift problem. Wild type hemoglobin stored at -70.degree. C.
 has not demonstrated a similar effect.
 Studies have shown that hemoglobin blood substitutes offer a number of
 difficulties as well as benefits. Hemoglobin is a powerful tool for oxygen
 delivery, but its use removes a tightly regulated protein from its native
 environment. One major problem for hemoglobin based blood substitutes
 occurs when oxygen in the heme iron dissociates as superoxide ion, leaving
 hemoglobin oxidized in the ferric "met" state. This autoxidation leaves
 hemoglobin in a state where it cannot bind ligand. Moreover, the Fe+++
 state is an intermediate in the pathway to the highly reactive Fe.sup.4 +
 ferryl state, heme loss, and can cause peroxidation of lipids (Giulivi,
 C., and Davies, K. J. A. (1994), Methods of Enzymology 231, 490-496;
 Yamamoto, Y., and La Mar, G. N. (1986) Biochemistry 25, 5288-5297; Repka,
 T., and Hebbel, R. P. (1991) Blood 78, 2753-2758). Superoxide off-rates
 appear to govern the measured autoxidation rate.
 Another key problem associated with hemoglobin-based blood substitutes is
 hemoglobin's affinity for nitric oxide (NO), which is higher than its
 affinity for CO or O.sub.2. NO is a vasodilator and can be carried by
 hemoglobin as a heme ligand or on a cysteine as a nitrosothiol
 (Bonaventura (1996) Nature 380, 221-226). Results in clinical trials
 demonstrate that patients treated with a hemoglobin-based blood substitute
 often encounter higher blood pressure (Blantz, R. C., Evan, A. P., and
 Gabbai, F. B. (1995) in Blood Substitutes: Physiological Basis of Efficacy
 (Winslow, R. M., Vandegriff, K. D., and Intaglietta, M., Ed.) pp. 132-142,
 Birkhauser, Boston). Another problem with hemoglobin is that the molecule
 is small enough to extravasate into the endothelial lining and bind NO.
 Patients treated with L-arginine, an intermediate in the NO synthesis
 pathway, or nitroglycerine, a vasodilator, have normal blood pressures
 while being administered hemoglobin solutions (see Blantz, R. C., Evan, A.
 P., and Gabbai, F. B. (1995) in Blood Substitutes: Physiological Basis of
 Efficacy, supra.
 Perhaps the most significant drawback of hemoglobin blood substitutes is
 the rapid filtration of hemoglobin molecules by the kidney. At
 concentrations used in patients, hemoglobin dissociates into alpha/beta
 dimers small enough for renal filtration. This not only significantly
 decreases the lifetime of the blood substitute (half life of less than an
 hour), but it also deleteriously effects renal tubules and can cause renal
 toxicity (see Blantz, R. C., Evan, A. P., and Gabbai, F. B. (1995) in
 Blood Substitutes: Physiological Basis of Efficacy, supra).
 One important step in eliminating renal toxicity is the cross-linking of
 alpha/beta dimers. Current efforts include chemical cross-linking of two
 alphas or two betas with a covalent attachment to lysine residues
 (Vandegriff, K. D., & Le Telier, Y. C. (1994)
 Artificial-Cells-Blood-Substitutes-and-Immobilization-Biotechnology 22,
 443-455). In addition, hemoglobins have been randomly polymerized using
 glyceraldehyde (Vandegriff, K. D., & Le Telier, Y. C. (1994)
 Artificial-Cells-Blood-Substitutes-and-Immobilization-Biotechnology 22,
 443-455). However, utilization of a chemical reaction significantly lowers
 the yield of functional protein.
 Researchers have produced a genetically cross-linked hemoglobin molecule
 with a half life of almost two hours (Looker, D., Abbott-Brown, D.,
 Cozart, P., Durfee, S., Hoffman, S. Mathews, A., Miller-Roehrich, J.,
 Shoemaker, S., Trimble, S., Fermi, G., Komiyama, N. H., Nagai, K., &
 Stetler, G. L. (1992) Nature 356, 258-260). X-ray crystallography has
 shown the C-terminus of one alpha chain to be only 2 to 6 .ANG. away from
 the N-terminus of the second alpha chain (Shaanan, B., (1983) Journal of
 Molecular Biology 171, 31-59), and trypsin catalyzed reverse hydrolysis
 has demonstrated that an additional amino acid attached to the C-terminus
 does not alter oxygen binding properties. These results, coupled with the
 knowledge that the C-terminal arg141 can form a salt bridge with the
 alternate alpha chain's vall, demonstrated the feasibility of genetically
 cross-linking the two alpha chains. The di-alpha chain expressed by these
 workers in E. coli consisted of an alpha des-val, a glycine linker, and a
 native alpha chain sequence. The construct was co-expressed with a des-val
 version of a naturally occurring low-oxygen affinity beta mutant (beta
 Presbyterian, R108K), and the entire construct was dubbed rHb1.1.
 Despite these extensive efforts to develop a hemoglobin-based blood
 substitute, needs still exist for substitutes with increased crosslinking
 and higher molecular weight, which provide increased molecular stability
 and plasma half-life, and a decreased risk of renal toxicity. Such
 substitutes will desirably be readily expressed in host cells in high
 yield and have advantageous oxygen-binding capacity. The present invention
 addresses these needs.
 SUMMARY OF THE INVENTION
 Accordingly, one preferred embodiment of the invention provides a heme
 protein which includes a (i.e. at least one) hemoglobin molecule including
 at least one circularly permuted globin. In a preferred form, the
 invention takes advantage of the close proximity of the N and C termini of
 neighboring alpha chains, and a linker of one or more amino acids is
 inserted between both sets of termini. New termini are formed at any
 sequence position in the protein, and preferably at a position so as to be
 surface-exposed for linkage with other molecules, for example one or more
 other hemoglobin molecules to form recombinant hemoglobin multimers.
 Preferred proteins of the invention include at least one oxygen-binding
 hemoglobin tetramer having two alpha and two beta globins, wherein at
 least one of the globins is circularly permuted, and, more preferably, has
 surface-exposed N- and C-termini. Still more preferably, the hemoglobin
 molecule(s) in proteins of the invention will have multiple crosslinks
 between globins.
 Another preferred embodiment of the invention provides a heme protein,
 preferably oxygen-binding, which includes at least one hemoglobin molecule
 including two beta globins and a di-alpha globin construct. The di-alpha
 globin construct includes a circularly-permuted alpha globin genetically
 crosslinked to another alpha globin. Thus the preferred di-alpha globin
 construct will include an amino acid sequence corresponding to a circular
 permutation of single polypeptide which has alpha chains whose original N-
 and C-termini are each linked to one another by a linker sequence of one
 or more amino acids. In an advantageous form, the di-alpha construct can
 be covalently linked to another protein, e.g. another di-alpha construct,
 by a polypeptide linker, to form proteins of high molecular weight, e.g.
 hemoglobin multimers.
 Another preferred embodiment of the present invention provides a
 polynucleotide coding for a single polypeptide having a
 circularly-permuted alpha globin covalently linked to another alpha globin
 by two genetic crosslinks. Thus, preferred polynucleotides will
 sequentially encode (1) a first portion of a first, circularly-permuted
 alpha globin; (2) a first genetic crosslink; (3) a second alpha globin;
 (4) a second genetic crosslink; and (5) a second portion of the circularly
 permuted alpha globin, the first and second portions together constituting
 the entire circularly-permuted alpha globin. Thus, preferred
 polynucleotides will code for two alpha globins, a first of which is
 circularly permuted and a second of which is non-circularly permuted and
 occurs in the polypeptide linking the original N- and C-termini of the
 first alpha globin.
 Still another preferred embodiment of the invention provides a
 circularly-permuted globin having termini located within a surface-exposed
 loop region of the globin (i.e. within any non-helicle surface-exposed
 alpha segment). The preferred, surface-exposed termini will be
 solvent-exposed (having no structures of the globin overlying the
 termini), and effective for covalent linking of one or both termini to an
 adjacent hemoglobin alpha or beta subunit, or to another molecule, e.g. to
 form a fusion protein. Preferred circularly-permuted alpha globins will
 have as terminal amino acids, residues 47 and 48, 48 and 49, 49 and 50, 50
 and 51, 113 and 114, 114 and 115, 115 and 116, or 116 and 117 of the
 corresponding non-circularly permuted globin. Preferred
 circularly-permuted beta globins will have as terminal amino acids,
 residues 46 and 47, 47 and 48, 48 and 49, 118 and 119, 119 and 120, 120
 and 121 and 121 and 122 of the non-circularly permuted beta globin.
 Other preferred embodiments of the invention provide a polynucleotide
 encoding a circularly-permuted globin, a vector or host cell including
 such a polynucleotide, and a method for preparing a heme protein which
 involves culturing a host cell including and expressing such a
 polynucleotide.
 The present invention also relates to a method of increasing tissue
 oxygenation in a warm blooded animal patient, e.g. human patient,
 comprising administering to the patient a therapeutically effective amount
 of an oxygen-binding heme protein of the invention.
 The present invention also provides a method of replacing hemoglobin in the
 bloodstream of a warm blooded animal patient, e.g., a human patient,
 comprising administering to the patient an effective amount of a heme
 protein of the invention.
 A still further preferred embodiment of the invention provides a method for
 inducing vasoconstriction in a warm blooded animal, e.g. a human patient,
 comprising introducing into the blood stream of the animal an effective
 amount of an oxygen-binding heme protein of the invention.
 Another preferred embodiment of the invention provides a method for
 increasing the oxygenation of an isolated organ or tissue, for example
 during storage or transport, which includes the step of contacting the
 organ or tissue with an oxygen-binding heme protein of the invention.
 Additional embodiments as well as objects, features and advantages of the
 invention will be apparent from the following description.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 For the purposes of promoting an understanding of the principles of the
 invention, reference will now be made to embodiments thereof and specific
 language will be used to describe the same. It will nevertheless be
 understood that no limitation of the scope of the invention is thereby
 intended, such alterations, further modifications and applications of the
 principles of the invention as described herein being contemplated as
 would normally occur to one skilled in the art to which the invention
 pertains.
 The following definitions are used herein.
 Nucleotide--A monomeric unit of DNA or RNA containing a sugar moiety
 (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is
 linked to the sugar moiety via the glycosidic carbon (1' carbon of the
 pentose) and that combination of base and sugar is called a nucleoside.
 The base characterizes the nucleotide. The four DNA bases are adenine
 ("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNA
 bases are A, G, C and uracil ("U").
 Polynucleotide--A linear array of nucleotides connected one to the other by
 phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
 Polypeptide--A linear array of amino acids connected one to the other by
 peptide bonds between the alpha-amino and carboxy groups of adjacent amino
 acids.
 Expression--The process undergone by a structural gene to produce a
 polypeptide. It is a combination of transcription and translation.
 Plasmid--A non-chromosomal double-stranded DNA sequence comprising an
 intact "replicon" such that the plasmid is replicated in a host cell.
 Vector--A plasmid, viral DNA or other DNA sequence which is capable of
 replicating in a host cell, which is characterized by one or a small
 number of endonuclease recognition sites at which such DNA sequences may
 be cut in a determinable fashion without attendant loss of an essential
 biological function of the DNA, e.g., replication, production of coat
 proteins or loss of promoter or binding sites, and which contains a marker
 suitable for use in the identification of transformed cells, e.g.,
 tetracycline resistance or ampicillin resistance
 Transformation--The introduction of DNA or RNA into cells in such a way as
 to allow gene expression.
 Stroma--free preparation--a preparation free from red blood cells and red
 blood cell membrane fragments.
 Crosslinked Hemoglobin Molecule--A hemoglobin molecule modified by covalent
 bond crosslinkage between one or more of its globin subunits.
 Circularly-Permuted Globin--A globin having a covalent bond linkage between
 its native terminal amino acid residues and which has new terminal amino
 acid residues at another location of its polypeptide chain.
 Genetic Crosslink--An amino acid or a polypeptide which covalently links
 two globins of a hemoglobin tetramer and which is formed upon expression
 of a polynucleotide encoding the two globins and the chain as a single
 polypeptide.
 Native Termini--The termini amino acid residues of a globin prior to its
 circular permutation.
 New Termini--The terminal amino acid residues of a globin after its
 circular permutation.
 Globin--A compact protein domain containing heme preferably capable of
 forming higher molecular weight aggregates.
 Hemoglobin--A protein of four globins.
 Circularly-Permuted Hemoglobin Multimer--A protein which includes two or
 more hemoglobin molecules each having at least one circularly-permuted
 globin, wherein the circularly-permuted globins of adjacent hemoglobin
 molecules are covalently linked to one another by a linker chain of one or
 more amino acids.
 As disclosed above, the present invention concerns novel heme proteins
 which have at least one hemoglobin molecule including at least one
 circularly-permuted globin. In this regard, a circularly permuted (CP)
 protein has its native termini linked, and new termini at some other
 location in its polypeptide chain. Thus, circularly-permuted proteins can
 be prepared by creating a circular primary sequence of the protein, and
 then recleaving the protein at another site, or by expression of a DNA
 sequence encoding an amino acid sequence corresponding to the recleaved
 protein. The resulting protein represents a new protein which has a
 primary amino acid sequence which differs significantly from that of the
 starting protein. However, for the sake of simplicity in nomenclature, the
 art has adopted the practice of referring to the new protein as a
 circularly-permuted "starting protein", a practice which will be followed
 herein for the sake of convenience when referring to a peptide having a
 primary amino acid sequence which corresponds to a circular permutation of
 the primary amino acid sequence of a known globin such as an alpha or beta
 globin. Similarly, tetrameric heme proteins of the invention which
 incorporate one or more circularly-permuted globins will be referred to as
 hemoglobins.
 The present invention can be applied to conventional human hemoglobin and a
 wide variety of known hemoglobin mutants. In this regard, the amino acid
 sequences for the alpha and beta globins of conventional human hemoglobin
 are provided in Table 2, in which the abbreviations in Table 1 are
 employed.
 TABLE 1
 Amino Acid Abbreviation
 Alanine Ala
 Arginine Arg
 Asparagine Asn
 Aspartic acid Asp
 Cysteine Cys
 Glutamine Gln
 Glutamic acid Glu
 Glycine Gly
 Histidine His
 Isoleucine Ile
 Leucine Leu
 Lysine Lys
 Methionine Met
 Phenylalanine Phe
 Proline Pro
 Serine Ser
 Threonine Thr
 Tryptophan Trp
 Tyrosine Tyr
 Valine Val
 TABLE 2
 Beta Globin Alpha Globin
 1 Val 1 Val
 2 His 2 Leu
 3 Leu 3 Ser
 4 Thr 4 Pro
 5 Pro 5 Ala
 6 Glu 6 Asp
 7 Glu 7 Lys
 8 Lys 8 Thr
 9 Ser 9 Asn
 10 Ala 10 Val
 11 Val 11 Lys
 12 Thr 12 Ala
 13 Ala 13 Ala
 14 Leu 14 Trp
 15 Trp 15 Gly
 16 Gly 16 Lys
 17 Lys 17 Val
 18 Val 18 Gly
 19 Asn 19 Ala
 20 Val 20 His
 21 Asp 21 Ala
 22 Glu 22 Gly
 23 Val 23 Glu
 24 Gly 24 Tyr
 25 Gly 25 Gly
 26 Glu 26 Ala
 27 Ala 27 Glu
 28 Leu 28 Ala
 29 Gly 29 Leu
 30 Arg 30 Glu
 31 Leu 31 Arg
 32 Leu 32 Met
 33 Val 33 Phe
 34 Val 34 Leu
 35 Tyr 35 Ser
 36 Pro 36 Phe
 37 Trp 37 Pro
 38 Thr 38 Thr
 39 Gln 39 Thr
 40 Arg 40 Lys
 41 Phe 41 Thr
 42 Phe 42 Tyr
 43 Glu 43 Phe
 44 Ser 44 Pro
 45 Phe 45 His
 46 Gly 46 Phe
 47 Asp 47 Asp
 48 Leu 48 Leu
 49 Ser 49 Ser
 50 Thr 50 His
 51 Pro 51 Gly
 52 Asp 52 Ser
 53 Ala 53 Ala
 54 Val 54 Gln
 55 Met 55 Val
 56 Gly 56 Lys
 57 Asn 57 Gly
 58 Pro 58 His
 59 Lys 59 Gly
 60 Val 60 Lys
 61 Lys 61 Lys
 62 Ala 62 Val
 63 His 63 Ala
 64 Gly 64 Asp
 65 Lys 65 Ala
 66 Lys 66 Leu
 67 Val 67 Thr
 68 Leu 68 Asn
 69 Gly 69 Ala
 70 Ala 70 Val
 71 Phe 71 Ala
 72 Ser 72 His
 73 Asp 73 Val
 74 Gly 74 Asp
 75 Leu 75 Asp
 76 Ala 76 Met
 77 His 77 Pro
 78 Leu 78 Asn
 79 Asp 79 Ala
 80 Asn 80 Leu
 81 Leu 81 Ser
 82 Lys 82 Ala
 83 Gly 83 Leu
 84 Thr 84 Ser
 85 Phe 85 Asp
 86 Ala 86 Leu
 87 Thr 87 His
 88 Leu 88 Ala
 89 Ser 89 His
 90 Glu 90 Lys
 91 Leu 91 Leu
 92 His 92 Arg
 93 Cys 93 Val
 94 Asp 94 Asp
 95 Lys 95 Pro
 96 Leu 96 Val
 97 His 97 Asn
 98 Val 98 Phe
 99 Asp 99 Lys
 100 Pro 100 Leu
 101 Glu 101 Leu
 102 Asn 102 Ser
 103 Phe 103 His
 104 Arg 104 Cys
 105 Leu 105 Leu
 106 Leu 106 Leu
 107 Gly 107 Val
 108 Asn 108 Thr
 109 Val 109 Leu
 110 Leu 110 Ala
 111 Val 111 Ala
 112 Cys 112 His
 113 Val 113 Leu
 114 Leu 114 Pro
 115 Ala 115 Ala
 116 His 116 Glu
 117 His 117 Phe
 118 Phe 118 Thr
 119 Gly 119 Pro
 120 Lys 120 Ala
 121 Glu 121 Val
 122 Phe 122 His
 123 Thr 123 Ala
 124 Pro 124 Ser
 125 Pro 125 Leu
 126 Val 126 Asp
 127 Gln 127 Lys
 128 Ala 128 Phe
 129 Ala 129 Leu
 130 Tyr 130 Ala
 131 Gln 131 Ser
 132 Lys 132 Val
 133 Val 133 Ser
 134 Val 134 Thr
 135 Ala 135 Val
 136 Gly 136 Leu
 137 Val 137 Thr
 138 Ala 138 Ser
 139 Asn 139 Lys
 140 Ala 140 Tyr
 141 Leu 141 Arg
 142 Ala
 143 His
 144 Lys
 145 Tyr
 146 His
 There are also hundreds of known mutations of hemoglobin which involve
 changes in the amino acid structure of the polypeptide chains. For
 example, a mutant form of alpha globin (des-val) is used in the specific
 Experimental below. This alpha globin has a valine.fwdarw.methionine
 substitution at amino acid 1 of the chain. Other known alpha mutants
 include but are not limited to such modifications as amino acid 94
 aspartic acid .fwdarw.asparigine in the alpha chain (Hb Titusville).
 A number of known mutant hemoglobins have amino acid substitutions at human
 beta globin positions 90, 102, 108 and combinations thereof. Some specific
 examples of beta mutations are but are not limited to:
 (1) amino acid 90 glutamine.fwdarw.lysine (hemoglobin Agenogi)
 (2) amino acid 90 glutamine.fwdarw.glycine
 (3) amino acid 108 asparagine.fwdarw.aspartic acid (hemoglobin Yoshizuka)
 (4) amino acid 102 asparagine.fwdarw.threonine (hemoglobin Kansas)
 (5) amino acid 102 asparagine.fwdarw.serine (hemoglobin Beth Israel)
 (6) amino acid 90 glutamic acid.fwdarw.valine amino acid 91
 Leucine.fwdarw.methionine amino acid 93 cysteine.fwdarw.serine amino acid
 94 aspartic.fwdarw.glutamic acid
 A Table including a listing of some additional illustrative hemoglobin
 variants is set forth in Appendix A attached hereto and made a part
 hereof, taken from Hemoglobin, Vol 19, No. 1-2, pp. 39-124, Marcel Dekker
 (1995).
 In addition to known mutations other mutations can be engineered into these
 circularly permuted globins in order to add additional desirable
 properties into the protein or protein multimer. For example, mutations
 that alter the electronic environment of the heme may be included to
 stabilize the reduced, physiologically active, form of the molecule or
 alter the ligand affinity and selectivity.
 Generally speaking, in the present invention, the new termini of the globin
 subunit are formed at a site that does not eliminate the function of the
 globin in assembling with other globins to form an oxygen-binding,
 tetrameric heme protein. Thus, the resulting protein will possess the
 function of interest of the wild type hemoglobin, e.g. the capacity to
 bind oxygen at some level, which can be the same level, or a level which
 is increased or decreased relative to the wild type protein.
 Generally speaking, preferred candidate locations for forming the new
 termini will fall within surface-exposed loop regions on the globins,
 rather than in alpha helical segments. This is expected to minimize
 disruption of the protein structure since the loops are not highly
 ordered. More preferred regions for introducing new termini in alpha
 globins include the loop region between the C and E helices (residues
 47-51) and the loop region between the G and H helices (residues 113-117).
 The loop region between the C and E helices is most preferred. Thus, in
 the Experimental below, new termini were created at original serine 49
 (new N terminus) and original leucine 48 (new C terminus) of normal adult
 human alpha globin.
 In the circular permutation of a human beta globin (see e.g. Table 1), a
 longer linker may be used to join the native termini because the termini
 are not as spacially close as those of the alpha chains. For example, a
 polypeptide linker of about three to five residues may be used. Because
 the beta chains are structurally similar to the alpha chains, the
 preferred sites for introduction of new termini in beta chains generally
 include the same loop regions selected for the alpha chains. These
 include, but are not limited to, the loop region between helices C and D
 (residues 46-49), and the loop region between helices G and H (residues
 118-122). Of these, the loop region between helices C and D is most
 preferred.
 The new termini of the circularly-permuted globins of the invention are
 preferably exposed on the surface of the globin when assembled in the
 ellipsoid, tetrameric hemoglobin. The selection of new termini may be
 assisted in this regard by conventional modeling software, for example
 modeling protein structure on a Silicon Graphics Imaging computer using
 molecular modeling software to verify surface exposure of amino acids.
 Location of the new termini at the surface of the hemoglobin molecule
 facilitates covalent linkage of the molecule to other molecules through
 amino acid or polypeptide linkers. In one preferred practice of the
 invention, a hemoglobin multimer is provided, in which a plurality of
 hemoglobin molecules are covalently linked to one another by polypeptide
 linkers spanning between circularly-permuted globins of the respective
 hemoglobin molecules. In this regard, the length of this polypeptide
 linker can vary widely to suit a particular application; however, it is
 expected that polypeptide linkers having about one to about twenty amino
 acids will be suitable for most applications, more commonly having about
 one to about ten amino acids. In the applicants' preferred work, the
 polypeptide linker included a number of glycine residues in order to
 impart conformational freedom to the linker. In addition, the
 intermolecular linker will likely be solvent exposed, and thus hydrophilic
 residues can be used to advantage, for example, amino acid residues
 containing hydroxyl or acidic groups, e.g. the hydroxyl-containing serine
 used in the specific work reported in the Experimental below. Generally
 speaking, the selection and use of suitable amino acids in the
 intermolecular linker will be well within the purview of those skilled in
 the field.
 Similarly, the number of hemoglobin molecules in hemoglobin multimers of
 the invention may vary, including multimers having up to and exceeding
 about one hundred hemoglobin repeating units. Again, for most applications
 it is expected that a smaller number of repeating units will be suitable,
 e.g. in the range of two to about ten hemoglobin repeating units.
 To create intramolecular crosslinks between globins of a hemoglobin
 molecule, it is desirable to use a chain of one to about seven amino
 acids, more preferably from one to about three amino acids. Any suitable
 amino acid or set of amino acids may be used for this purpose, including
 for example one or more amino acids selected from those identified in
 Table 1, above. The selection and use of suitable amino acids in the
 crosslinks will be well within the purview of those skilled in the field.
 Amino acid crosslinks such as those discussed above are conveniently
 introduced as genetic crosslinks. Other modes of introducing
 intramolecular and/or intermolecular crosslinks may also be used,
 including for example chemical treatment with crosslinking agents. Such
 crosslinking agents may include dialdehydes, such as glyoxal, amlonic
 dialdehyde, succinic dialdehyde, glutaraldehyde, adipaldehyde,
 3-methylglutaraldehyde, propyladipaldehyde, phthalic dialdehyde,
 terephthaldehyde and malonic dialdehyde. See, e.g. Bonsen et al., U.S.
 Pat. Nos. 4,001,200; 4,001,401; and 4,053,590; and Bonhard et al., U.S.
 Pat. Nos. 4,136,093 and 4,336,248.
 Preferred crosslinked hemoglobin molecules will exhibit increased molecular
 stability as compared to native, non-crosslinked hemoglobins. This
 stability may be demonstrated, for instance, by increased thermal
 stability (e.g. melting points) of the hemoglobin molecules as compared to
 their non-crosslinked counterparts.
 The present invention also concerns an isolated polynucleotide, preferably
 DNA sequence, coding for a circularly-permuted globin, such as a human
 alpha or human beta globin. Such polynucleotides can be created, for
 example, by chemical synthesis of a polynucleotide having the desired
 sequence corresponding to a circular permutation of the globin gene at
 hand. Such polynucleotides of the invention may also be prepared by
 ligating the ends of the globin coding sequence of interest, directly or
 via a base sequence coding for an amino acid linker, and then cleaving the
 resulting circular sequence to result in the circularly-permuted sequence
 Genetic manipulations to create circularly-permuted sequences can be
 applied without undue experimentation to form a wide variety of
 polynucleotides coding for circularly-permuted globins and constructs
 including them, in accordance with the present invention.
 In a preferred aspect the invention provides a polynucleotide, such as a
 DNA or RNA sequence, preferably a DNA sequence, which encodes a single
 polypeptide which includes, in sequence: (I) a first portion of a first,
 circularly-permuted globin; (ii) a first genetic crosslink; (iii) a
 second, entire globin; (iv) a second genetic crosslink; and (v) a second
 portion of the circularly-permuted globin, wherein the first and second
 portions together constitute the entire circularly-permuted globin. The
 coded polypeptides also form a part of the present invention and,
 generally speaking, include a first, circularly-permuted globin having its
 original N- and C-termini joined by a linking polypeptide including the
 amino acid sequence of a second, non-circularly-permuted alpha globin.
 More preferably, the first and second globins of such constructs are alpha
 globins, and the first and second genetic crosslinks (the peptide
 sequences occurring between the N- and C-termini of the circularly
 permuted globin and those of the non-circularly-permuted globin) will have
 from one to about three amino acids.
 DNA or other polynucleotides for use in carrying out the present invention
 may be synthetically created, by hand or with automated apparatus. Means
 for synthetic creation of the polynucleotide sequences of the invention
 are generally known to those of ordinary skill in the art, particularly in
 light of the teachings contained herein. For additional details as to
 polynucleotide synthesis, reference can be made to standard texts on the
 subject including for instance Maniatis et al., Molecular Cloning- A
 Laboratory Manual, Cold Spring Harbor Laboratory (1984), and Horvath et
 al. An Automated DNA Synthesizer Employing Deoxynucleoside
 3'-Phosphoramidites, Methods in Enzymology 154:313-326, 1987, both hereby
 incorporated herein by reference. Additionally, polynucleotide sequences
 of the invention may be constructed by isolating and modifying a
 polynucleotide which occurs in nature. For instance, a starting globin
 polynucleotide may be a restriction fragment isolated from a genomic or
 cDNA library. The starting polynucleotide can then be manipulated using
 known techniques to produce a polynucleotide of the invention which
 encodes a circularly-permuted globin, generally as discussed above.
 The invention also provides expression or cloning vectors including
 polynucleotide sequences of the invention, including for instance plasmid
 vectors, viral vectors, and the like. The synthesis and/or isolation of
 necessary and desired component parts of expression or cloning vectors,
 and their assembly, is within the abilities of those of ordinary skill in
 the art and, as such, are capable of being performed without undue
 experimentation.
 The present invention also concerns a host cell including a polynucleotide
 of the invention and which expresses the polynucleotide. Such host cells
 can be made by transforming a cell with a suitable vector carrying a
 polynucleotide of the invention, for example a plasmid or viral vector.
 The polynucleotide of the invention can also be introduced into cells
 using other known techniques, including for example microinjection,
 electroporation or the like.
 The host cell can be selected from a variety of host cells which
 effectively express hemoglobin, including for instance mammalian cells
 such as human, murine or porcine cells, gram positive or negative
 bacterial cells such as E. Coli., Bacillus or Salmonella, yeast cells such
 as Sacharomyces Cerevisiae or Sacharomyces Pombe, or insect cells.
 Further, host cells which express polynucleotides of the invention can be
 cultured so as to produce circularly-permuted globins of the invention in
 high yield. The globins can then be individually isolated or, more
 preferably, the circularly-permuted globin is co-expressed in the host
 cell with other globins as necessary to produce the oxygen-binding heme
 protein including at least one assembled hemoglobin tetramer in the cell.
 Thus, for instance, in the Experimental below, the di-alpha or tetra-alpha
 globin constructs were co-expressed in host cells with beta globin, and
 the corresponding assembled hemoglobin tetramer or octamer were isolated
 from the cells in high yield. In this regard, isolation and purification
 of inventive proteins from the cultured host cells can be achieved using
 conventional techniques such as filtration, centrifugation,
 chromatography, and the like. Substantially purified preparations of heme
 proteins of the invention can thereby be prepared.
 Heme proteins of the invention exhibit useful properties as blood and
 hemoglobin substitutes. For example, the tetrameric and octameric heme
 proteins disclosed in the Experimental exhibit increased stability against
 thermal denaturation as compared to prior-known hemoglobin-based blood
 substitutes. Also, ligand binding experiments have demonstrated that these
 proteins possess ligand binding properties characteristic of wild-type
 hemoglobin, including oxygen binding, geminate recombination and CO
 on-rate.
 For use, heme proteins can be incorporated into pharmaceutically acceptable
 carriers to form pharmaceutical compositions, if desired. Sterile, liquid
 carriers, particularly aqueous carriers, or liposomes or other
 polymerizing and encapsulating polymers, will be preferred, for example a
 balanced electrolyte and buffer solution. The heme protein is desirably at
 a concentration of about 1 to about 20% in solution, with the precise
 concentration employed depending upon the application. The hemoglobin may
 also be dissolved in known plasma expanders such as colloids (plasma,
 albumin) or crystalloids (saline, glucose, dextran, gelatins, Hemasol* or
 Lactated Ringer's), or contained in natural red blood cells or in
 artificial red blood cells such as liposomes.
 The thus-prepared pharmaceutical preparation can then be conventionally
 administered to a human or other animal patient, for example by injection,
 catheterization, or the like. For convenience in these purposes, the
 pharmaceutical preparation can be contained in a sterile, medical-grade
 container such as a vial, syringe, or infusion bag.
 The oxygen-binding heme proteins of the invention may be used, for
 instance, in a stroma-free hemoglobin-type blood replacement, or to
 improve tissue oxygenation in disease states associated with compromised
 oxygen delivery to tissue including myocardial infarction, stroke, small
 vessel disease such as diabetes, etc. Heme proteins of the invention can
 further be used to increase oxygenation of tissues (e.g. tumor cells or
 other tissues having hypoxic cells due to damage by physical or chemical
 means, e.g., burns, exposure to chemicals, physical injuries or ionizing
 radiation). In one specific application, heme proteins of the invention
 may be used to increase oxygenation in hypoxic tumor cells to be subjected
 to radiation therapy, so as to increase the efficacy of the therapy (see,
 e.g., U.S. Pat. No. 5,295,944). Many tumors exhibit oxygen heterogeneity,
 including regions of hypoxia, which protect tumor cells against the
 cytotoxic action of ionizing radiation. Examples include solid tumors such
 as sarcomas, carcinomas and lymphomas, and some cases of dispersed tumor
 cells wherein masses of tumor cells form which can produce regions of
 oxygen heterogeneity, e.g. advanced leukemia. In such cases an increase in
 the oxygenation of the tumor tissue can enhance the effect of radiation
 therapy on the tissue.
 In order to increase oxygen transport to the site of a tumor, a preparation
 including a heme binding protein of the invention can be administered,
 e.g. intravenously, to the patient. The chemotherapeutic agent can then be
 administered, with the amount of time between the administration of the
 heme protein preparation and chemotherapeutic agent depending upon factors
 such as the amount of time it takes the heme protein preparation to be
 fully incorporated into the circulatory system of the host, the lifetime
 of the preparation, etc. Also, the patient may breath oxygen-enriched gas
 prior to and after the administration of the ionizing radiation. This can
 be done by having the host breath oxygen-enriched air, 100% oxygen or
 carbogen (95% oxygen/5% CO.sub.2), or in certain cases exposing the host
 to hyperbaric oxygen conditions.
 Any type of ionizing radiation which exhibits an antitumor effect can be
 employed, including as examples X-rays, gamma rays, high-energy electrons
 and High LET radiation, such as protons, neurons and alpha particles. Such
 ionizing radiation can be administered using techniques well-known to
 those skilled in the art. For example, X-rays and gamma rays are applied
 by external and/or interstitial means from linear accelerators or
 radioactive sources. High energy electrons can be produced by linear
 accelerators. High LET radiation is also produced by linear accelerators
 and can also be applied from radioactive sources implanted interstitially.
 Dosages of the ionizing radiation are generally those conventionally
 applied in radiotherapeutic treatment of tumors, although in certain cases
 usage of the oxygen-binding heme protein may lower the necessary dosage of
 ionizing radiation.
 In another area, heme proteins of the invention can be used in low doses to
 increase perfusion when desired. For example, the heme protein may be
 administered to increase blood pressure from abnormally low levels, as in
 shock of hemorrhagic, cardiogenic or septic origin, or to increase blood
 pressure from normal levels to effect improved perfusion, for instance as
 in stroke therapy. Heme proteins of the invention may also be used in
 oxygen sensors.
 In addition, heme proteins of the invention may be attached, e.g.
 covalently bonded, to other molecules via a surface-exposed, terminal
 amino acid or a polypeptide extending therefrom, to form additional
 materials in accordance with the present invention. For example, in one
 aspect, a heme protein of the invention can be conjugated to another
 molecule to form an active conjugate with increased vascular retention
 time as compared to that of the other molecule, and thus effectively
 modulate (increase) the vascular retention time of the other molecule. In
 one mode, the conjugate can be prepared by genetically linking a heme
 protein of the invention with the other molecule. Thus, a polynucleotide
 coding for both the heme protein and the other molecule can be constructed
 and introduced into a suitable host cell for expression. Expression of the
 DNA will then provide the conjugate. Heme proteins of the invention having
 surface-exposed termini will be particularly advantageous for these
 purposes. Because the termini are surface-exposed and not integrally
 involved in the structure of the heme protein, the linkage to other
 molecules will not significantly disrupt the structure of the heme
 protein, thus leaving it functional, and will also allow the attached
 molecule to remain in solution as opposed to being buried within the heme
 protein. There are a number of therapeutic peptides currently in use which
 would be expected to benefit from increased retention times when
 conjugated with heme proteins of the invention, including for instance
 insulin, erythropoietin, and growth hormones such as somatotropins. In the
 case of erythropoietin, as an example, the administration of a heme
 protein of the invention genetically linked to erythropoietin (a hormone
 which promotes red blood cell production in the body), may be used to
 replenish red blood cell supply of a patient before the blood substitute
 has degraded and been filtered by the blood stream.
 Heme proteins of the invention can also be attached ex-vivo to non-peptides
 such as organic molecules, DNA and the like. Numerous methods are known
 for covalently linking compounds to specific chemical moieties on
 proteins, such as directing crosslinking reagents to lysine on heme
 proteins of the invention provide a unique reactive sites on the protein
 for crosslinking to other molecules, which can be capitalized upon using
 known chemistries. Illustrative attachment chemistries are described for
 example in T. E. Creighton (1983) "Proteins: Structure and Molecular
 Properties", W. H. Freeman, N.Y.; and W. D. Dandliker and A. J. Portman
 (1971) "Excited States of Proteins and Nucleic Acids", R. F. Steiner and
 I. Weinryb eds., Plenum Press, N.Y., pp. 199-276.
 Heme proteins of the invention can also be attached to pharmaceutically
 active compounds in a specific 1:1 stoichiometry and it is expected that
 such will be accomplished without deleterious effect on the structural
 integrity of the heme proteins. Proteins of the invention may also be
 attached to targeting agents such as antibodies, or can be used
 advantageously as MRI imaging agents themselves or attached to other
 imaging (e.g. MRI or X-ray) or therapeutic agents.
 For the purposes of promoting a further understanding of the present
 invention, its principles and its advantages, the following Experimental
 is provided. It will be understood that this Experimental is illustrative,
 and not limiting, of the invention.
 EXPERIMENTAL
 E. coli strains and plasmid vectors used
 The E. coli strain DH5a was used in all the genetic engineering and protein
 expression of the gene constructs described below. Plasmids pHS471 and
 pWHS486, used in the genetic constructions, were generated previously by
 Hernan et. al. (Biochemistry, 31:8619-28 (1992)). pUC18 is a commercially
 available plasmid from New England BioLabs.
 Generation of DNA cassettes for genetic engineering
 Most of the genetic manipulations utilized cassette mutagenisis, which
 entails the generation of a pair of complementary oligonucleotides which
 contain the desired coding region. All oligonucleotides were synthetically
 prepared by and purchased from the genetic engineering facility at the
 University of Illinois. 400 picomoles of each oligonucleotide was
 phosphorylated with 10 units of T4 polynucleotide kinase in 100 .mu.L
 containing 50 mM Tris-HCl, pH 7.6/10 mM MgCl.sub.2 /5 mM DTT/100 .mu.M
 EDTA for 60 min. at 37.degree. C. After phosphorylation, the complementary
 oligonucleotides were annealled by mixing and heating to 95.degree. C. for
 5 min. and allowed to slowly cool to room temperature over a 3-4 hr.
 period. The resulting fragments have sticky ends corresponding to
 restriction sites allowing for ligation to gene fragments or plasmid
 vectors. For the purposes herein and as generally understood in the art, a
 "cassette" refers to a pair of complementary oligonucleotides prepared in
 the fashion described above.
 Plasmid Mini-Preparations
 All plasmid isolations were performed from a 5 mL overnight culture of E.
 coli DH5a, harboring the plasmid of interest, grown in LB media (10 g
 Tryptone, 5 g Yeast Extract, 5 g NaCl per liter). The commercially
 available Qiagen Spin Plasmid Mini-Preparation kit was used to purify the
 DNA. The Qiagen kit gives a high yield of RNA free plasmid. DNA were
 eluted in 25 .mu.L of water.
 Restriction digests
 All restriction digests were performed at 37.degree. C. 1-10 units of
 enzyme were used for each digestion and the reactions were carried out in
 a final volume of 10 .mu.L. The enzymes were purchased from New England
 Bio-Labs or GIBCO BRL. The buffer systems used were supplied with the
 enzymes.
 DNA fragment isolation and purification
 Restriction enzyme digested DNA fragments that were subsequently used in
 ligation reactions were separated on a 1% agarose gel with 10 .mu.g/mL
 ethidium bromide at 130 V for about 1 hour. The selected DNA bands were
 removed with a razor blade, and the resulting DNA was isolated from the
 gel fragment using the GeneClean II kit from BioLab 101.
 DNA Ligations
 Genecleaned plasmid fragments and oligonucleotides were ligated in a total
 reaction volume of 20 .mu.L with 1 unit of T4 DNA ligase(GIBCO BRL).
 Vector to fragment/cassette insert ratios varied between 1:3 and 1:50.
 Ligations ran for one hour at room temperature or 16.degree. C. overnight.
 Competent Cells and Transformations
 E. coli DH5a was grown in a 5 mL overnight culture of LB media. 500 .mu.L
 of the overnight culture was used to inoculate 50 .mu.L of LB. The cells
 grew 3-4 hours and were spun at 5000 g for 5 minutes. The cells were
 resuspended in 25 mL of cold 0.1 M CaCl.sub.2. After a 20 minute
 incubation on ice, the cells were again spun down under the same
 conditions. The pellet was then resuspended in 4 mL of cold CaCl.sub.2.
 Competent E. coli were transformed with finished ligation reactions or
 mini-prepped DNA. The ligation mixture or 0.5 .mu.g of mini-prepped DNA
 were mixed with 200 .mu.L of competent cells and incubated on ice for 20
 minutes. The cells were heat shocked at 37.degree. C. for 2 minutes and
 placed immediately on ice for 5 minutes. One mL of LB media was added, and
 the cells were incubated for one hour at 37.degree. C. in order to produce
 ampicillin resistance. 200 .mu.L of cells were plated on LB plates with
 agarose (15 g/L) and ampicillin (0.2 g/L). The remaining cell mixtures
 were spun down, part of the supernatant was removed, and the pelleted
 cells were resuspended and plated as well.
 DNA Sequencing
 Gene sequencing was performed at the University of Illinois DNA Sequencing
 Facility using a Perkin/Elmer DNA Sequencer with PCR amplification.
 Sequencing reactions were stopped with fluorescently labeled dideoxy
 nucleotides. Universal and reverse primers for pUC sequencing initiated
 the reactions.
 Construction of Di-alpha globin gene
 The alpha des-val gene (pHS471) was used to create a tandem fusion of two
 alpha globin gene sequences. pHS471 was digested with SalI and PstI to
 yield a vector containing the majority of the first alpha globin. Then an
 alpha gene fragment was generated from the digestion of pHS471 with SacI
 and PstI. This fragment was then ligated into the vector along with a
 linking cassette which codes for the last portion of the first alpha gene
 beginning with the SalI restriction site, a glycine codon, and the first
 portion of the second alpha gene through the SacI restriction site to
 generate pSS1. A schematic of this ligation procedure is included in FIG.
 1. The DNA sequence of the linking cassette and the di-alpha gene in pSS1
 is in FIGS. 2 and 3, respectively.
 The sequence of the construct was confirmed. The linking region was
 sub-cloned by digestion of the dialpha vector with BamHI and subcloning
 into the BamHI site of pUC18. The resulting plasmid was sequenced at the
 University of Illinois sequencing facility.
 Construction of the circularly permuted di-alpha gene
 The pSS1 created as described above was utilized for the generation of a
 circularly permuted dialpha globin gene. Two ligation steps and two
 cassettes were required for the synthesis. The sequence of cassette CP1
 and CP2 are shown in FIG. 4. In the first step, cassette CP1 and a 400
 base pair fragment generated from an MluI/BamHI digest of pSS1 was ligated
 into pHS471 digested with XbaI and BamHI to generate pSLS21 (FIG. 5). Then
 cassette CP2 and a 400 base pair fragment generated from an XhoI/BamHI
 digest of PSS1 was ligated into pSS21 digested with BamHI and PstI to
 generate pSLS22 (FIG. 6).
 The DNA sequence of pSLS21 was confirmed by sequencing in both the forward
 and reverse directions at the University of Illinois sequencing facility.
 The sequence along with the corresponding amino acids are shown in FIG. 7.
 A single operon was then created consisting of both circularly permuted
 di-alpha and desVal beta globin gene under the control of a single lac
 promoter. The circularly permuted di-alpha globin gene, isolated by
 digestion of pSLS22 with XbaI and PstI, and the beta globin gene, isolated
 by digestion of pWHS486 with PstI and HindIII, were ligated into pUC18
 digested with XbaI and HindIII to generate pSLS23 (FIG. 8).
 Construction of the tetra-alpha gene fusion
 Two circularly permuted di-alpha genes were fused with a linking region to
 create a tetra-alpha gene. This construct when expressed with beta globin
 genes forms a functionally octomeric globin consisting of 1 tetra-alpha
 globin and 4 beta globin genes. Three ligation steps and 2 cassettes were
 required for the creation of the tetra-alpha, beta globin gene operon.
 Cassettes used in the construction TA1 and TA2 are shown in FIG. 9. First,
 the StyI/BamHI fragment of pSLS22 and cassette TAl was ligated into pSLS22
 digested with XbaI and BamHI in order to destroy the StyI site of pSLS22,
 generating pSLS24(FIG. 10). Secondly, a 400 b.p. BamHI/XhoI fragment of
 pSLS22 and cassette TA2 was ligated into pSLS24 digested with BamHI and
 PstI generating pSLS25(FIG. 11). Finally, the XbaI/StyI fragment of
 pSLS25, the StyI/PstI fragment of pSLS22, and the PstI/HindIII fragment of
 pWHS486 was ligated into pUC18 digested with XbaI and HindIII generating
 pSLS26(FIG. 12). The DNA sequence which was confirmed by sequencing is
 listed in FIG. 13. The gene product of this construct will be hereafter
 referred to as octomeric circularly permuted hemoglobin.
 Construction of higher order circularly permuted alpha gene fusions
 To generate 6 fused alpha chain genes, 2 ligation steps are required as
 well as the utilization of a partial digest. First, pSLS26 is digested
 fully with PstI. The resulting linear DNA vector is then partially
 digested with BamHI using 3 units of enzyme in a non-ideal buffer for 15
 minutes. The 1350 b.p. fragment from this reaction is then isolated and
 ligated into pSLS22 digested with BamHI and PstI to generate pSLS27(FIG.
 14). Then pSLS27 is digested fully with PstI and partially digested, in a
 similar manner to the previous partial digest, with StyI. The 1500 b.p.
 fragment from this reaction is ligated into pSLS26 digested with StyI and
 PstI generating pSLS28(FIG. 15). In a similar manner any number of higher
 order alpha gene fusions can be created by the sequential ligation of
 circularly permuted dialpha segments from StyI partial and PstI digests of
 pSLS27 into progressively larger alpha gene fusion constructs.
 Expression of genetically engineered, novel heme proteins
 Plasmids pSLS23 and pSLS26 were transformed into competent E. coli DH5a
 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 .mu.g/mL ampicillin and 0.5
 mM d-aminolevulinic acid in 6 L shake flasks at 37.degree. C. After 36-48
 hours, the cells were harvested by centrifugation at 8000 g for 5 minutes
 and the cell paste was removed and stored frozen at -70.degree. C.
 Whole cell CO difference spectra
 To assay protein expression, a Carbon Monoxide (CO) difference spectrum was
 taken of the E. coli cell cultures before protein purification. Cells were
 grown to stationary phase in the conditions described for protein
 expression above. A few grains of dithionite were added to 3-5 mL of the
 culture, and a baseline was recorded. CO was then bubbled through the
 culture and the spectrum was recorded on a Cary13 spectrophotometer.
 Isolation and purification of protein
 Cell paste was allowed to thaw over a stream of CO, and all buffers used
 during the lysis and purification procedure were saturated with CO. Thawed
 cells were resuspended in 5 times (w/v) 10 mM NaH.sub.2 PO.sub.4, pH 6.0,
 1 mM EDTA. Cells were lysed by 3-4 passes through a Stansted AO-116 cell
 disrupter. After cell lysis 80units/mL DNase and 8 units/mL RNase were
 added to the mixture and allowed to incubate at room temperature for 1 hr.
 The mixture was then centrifuged at 100,000 g in a Beckman L8-M
 ultracentrifuge. The supernatant was retained and the pH adjusted to 6.0
 with 20 mM NaH.sub.2 PO.sub.4. The supernatant was then loaded onto a
 carboxy methyl cellulose column (Whatman) equilibrated with 10 mM
 NaH.sub.2 PO.sub.4, pH 6.0, 1 mM EDTA. The column was then washed with 4
 column volumes of 10 mM NaH.sub.2 PO.sub.4, pH 6.0, 1 mM EDTA, and finally
 eluted with a step gradient to 20 mM Tris-HCl, pH 7.0, 1 mM EDTA. The
 protein was then collected and concentrated using a PM-30 amicon membrane
 under nitrogen pressure. The concentrated protein was then flash frozen in
 liquid nitrogen and stored at -70.degree. C. until used.
 Mass spectrometry
 Purified protein samples were run over a sephadex G-25 column to exchange
 the protein into water. Electrospray mass spectrometry was performed on
 the protein samples at the University of Illinois Mass Spectrometry
 Facility. The samples were diluted to a concentration of 10 pmol/.mu.L
 into a 50:50 acetonitrile:water solution containing 0.1% formic acid for
 the experiments.
 The experimentally measured masses of the circularly permuted hemoglobin
 (CpHb) and octameric circularly permuted hemoglobin (OHb) were compared
 with calculated values. The beta globins of each protein was measured to
 be 15900.+-.2 Daltons. This is in good agreement with the calculated value
 of 15901.4 Daltons. The circularly permuted alpha globin construct of each
 protein are listed below. However, the calculated values are those
 expected for cleavage of the initial methionine residue. The values are in
 good agreement indicating that the proteins do not undergo any
 post-translational modification, except for initial methionine cleavage,
 in the bacterial host.

ABSORBANCE MAXIMA
 DEOXY Hb --OXY Hb --CO Hb
 Native Hb 430 555 414 541 576 419 539 568
 CPHb 429 555 414 540 577 419 538 568
 OHb 429 554 414 541 576 419 539 568
 SIS-PAGE
 Polyacrylamide gel electrophoresis was performed on the purified protein
 samples. Each protein sample was boiled for 5 minutes in 10 mM tris-CHl,
 pH 8.0, 1 mM EDTA, 2.5% SDS, 5% b-mercaptoethanol, and 0.001%
 bromophenol-blue. 2 .mu.L of the samples were loaded onto a 10-15%
 preformed gradient gel (Pharmacia). The gels were run using the Pharmacia
 PhastSystem for a total of 60 Volt hours at 250 Volts, 10.0 mA, 3.0 Watts,
 and 15.degree. C. The gels were developed in the PhastSystem developing
 chamber using the fast coomassie staining technique.
 Oxygen Equilibrium Measurements
 The oxygen equilibrium measurements taken in a homemade hemeox analyzer
 (Ron Hernan, Ph.D. Thesis, University of Illinois at Urbana Champaign,
 1994) are shown in FIG. 17. The equilibrium binding curves of native
 hemoglobin, circularly permuted hemoglobin (CpHb), and octomeric
 circularly permuted hemoglobin (OHb) in 0.05 M Tris-HCl buffer at pH 7.4
 with 0.1M [Cl.sup.- ] were performed at 60 .mu.M, 5 .mu.M, and 5 .mu.M in
 [heme] respectively. P.sub.50 values and n.sub.max values were then
 calculated for each protein. A bohr effect was determined by measure
 p.sub.50 values for each protein at pH 6.5 and 8.5 using 0.05 M Bis Tris
 with 0.1M [Cl.sup.- ] and 0.05M Tris-CHl with 0.1M [Cl.sup.- ]
 respectively. Finally each protein's response to allosteric effectors was
 measured with the addition of 0.1 mM IHP. Comparisons between the proteins
 are listed below and indicate that the circularly permuted hemoglobins
 cooperatively bind oxygen (n.sub.max =2) and that they still respond to
 allosteric effectors (protons and IHP) in a similar manner to native
 hemoglobin.

HbA.sub.o CpHb Octamer Hb
 P.sub.50 (mmHg) 5.0 .+-. 0.2 0.9 .+-. 0.1 0.8 .+-. 0.1
 N.sub.max 3.0 2.0 1.9
 .DELTA.logp.sub.50 .+-. 0.1 mM IHP 0.9 0.7 0.7
 Bohr Effect -0.5 -0.3 -0.4
 Plasma Lifetime Measurements
 Intravascular lifetime of the circularly permuted hemoglobin was measured
 and compared with the lifetime of cell free HbA. HbA was obtained from
 purification of freshly drawn blood as previously described (Ron Hernan,
 Ph.D. Thesis, University of Illinois at Urbana Champaign, 1994). Young
 adult Spaugue Dawley rats, weighing between 150 and 200 grams were used
 for the experiments. The protein samples were exchanged into a buffer
 solution consisting of 150 mM NaCl, 5 mM KCl, 2 mM NaPO.sub.4, at pH 7.4
 using a Sephadex G-25 column. The samples were then concentrated to
 approximately 1.5 mM in heme. 1 mL of this solution was filter sterilized
 and injected into each rat through a surgically placed jugular catheter.
 200 .mu.L blood samples were withdrawn from the same catheter at 5 minutes
 as a baseline and at successive time intervals. Plasma was separated from
 the erythrocytes by centrifugation, and the hemoglobin content was
 determined with the Plasma Hemoglobin Diagnostic Kit from Sigma. The
 measurements were then normalized to the 5 minute value and three
 experiments in three different rats were averaged for each protein.
 Exponential fits of the data are shown in FIG. 18, and the T.sub.1/2 were
 calculated to be 30 and 90 minutes for HbA and circularly permuted
 hemoglobin respectively.
 APPENDIX A