DNAs encoding genetically engineered low oxygen affinity mutants of human hemoglobin

A substantially pure polynucleotide coding for an alpha and/or beta human globin, the globin when part of a hemoglobin molecule confering lower oxygen affinity than normal hemoglobin to result in a mutant hemoglobin, the mutant hemoglobin having an oxygen affinity measured for stripped hemoglobin characterized by a P.sub.50 of 30 torr to 3 atmospheres and/or by a Hill coefficient between 2.5 and 1.0. Such mutant hemoglobin being useful to increase tissue oxygenation in a patient, to replace hemoglobin in the bloodstream of a patient and to treat patients suffering from burns.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention concerns genetically engineered low oxygen affinity 
mutants of human hemoglobin and the use of the same to increase tissue 
oxygenation in a human patient or to replace hemoglobin in a human 
patient. One aspect of the present invention resides in providing more 
oxygen to tumors. 
2. Background Information 
Blood consists of plasma and cells floating within it. The cells comprise 
white blood cells (leukocytes), platelets and red blood cells 
(erythrocytes). Red blood cells contain a protein, hemoglobin, which 
imparts color. It is the hemoglobin (Hb) which is involved in the 
transport of oxygen and carbon dioxide and which plays a role in 
regulating blood pH. 
Each molecule of hemoglobin comprises four smaller subunits, called 
polypeptide chains. These are the protein or globin parts of hemoglobin. A 
heme group, which is an iron-protoporphyrin complex, is associated with 
each polypeptide subunit and is responsible for the reversible binding of 
one molecule of oxygen. Normal adult hemoglobin is made up of two 
different kinds of polypeptide subunits. One is called the alpha chain 
containing 141 amino acid residues and the other is called the beta chain 
and contains 146 amino acid residues. Two of each kind of such polypeptide 
chains are arranged in the form of a truncated tetrahedron which has an 
overall shape of an ellipsoid. 
There are almost 300 known mutations of hemoglobin. Mutations lead to 
changes in the amino acid structure of the polypeptide chains. The 
abnormal gene responsible for sickle-cell anemia causes the normal 
glutamic amino acid residue at position six of the beta chain to be 
substituted by a valine group. Few of the known hemoglobin mutants, 
however, cause disease. Most mutants contain a mixture of mutant and 
normal hemoglobin. 
The reversible combination of hemoglobin and oxygen is represented by the 
following reaction: 
##STR1## 
The equilibrium constants for each step are not the same because an oxygen 
molecule on one heme group changes (increases) the affinity of the other 
hemes for additional oxygen molecules. This alteration in binding affinity 
during oxygenation is called heme-heme interaction or cooperativity. 
Because of this cooperativity, the relationship between the partial 
pressure of oxygen and the amount of oxygen bound to hemoglobin is 
represented by a sigmoid curve. It is customary to characterize this 
sigmoid curve by two parameters: P.sub.50, the partial pressure of oxygen 
at which half saturation of the hemoglobin takes place; and n, the 
so-called Hill coefficient, which is a measure of cooperativity and thus 
the sigmoid shape of the curve. The Hill coefficient can vary in value 
from 1.0, corresponding to a lack of cooperativity and a straight line 
relationship, to 4.0, maximum cooperativity and an extreme sigmoid shape. 
The oxygen affinity of hemoglobin is influenced by multiple factors 
including pH and the presence of certain inorganic phosphate molecules, 
most importantly 2,3-diphosphoglycerate. The term stripped hemoglobin is 
used to refer to hemoglobin free of these modulating inorganic phosphates. 
Stripped normal human hemoglobin has a P.sub.50 value of approximately 10 
torr and a Hill coefficient of 2.8 to 3.0. The decrease in oxygen 
affinity with decrease in pH (more acidic) is known as the Bohr effect. An 
extreme form of the Bohr effect, known as the Root effect is observed for 
certain fish hemoglobin for which the value of P.sub.50 at pH circa 6.5 
may be as high as several atmospheres. 
X-irradiation is an important modality in the treatment of solid tumors. 
Two-thirds of the biological damage produced by x-rays occurs indirectly 
and is mediated by the action of free radicals. Oxygen combines with the 
free radicals and "fixes" the lesion in the cell. Therefore a thoroughly 
oxygenated tumor will be more responsive to x-irradiation. 
Most solid tumors are not uniformly oxygenated. About 10-20% of the cells 
in experimental tumors are hypoxic. This occurs, at least in part, because 
tumors outgrow their blood supply. If more oxygen could be delivered to a 
tumor, cell death would be greater and tumor curability would improve. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide genetically engineered 
low oxygen affinity mutants of human hemoglobin. 
It is another object of the invention to provide a method of increasing 
tissue oxygenation in a human patient. 
It is a further object of the invention to provide a method of replacing 
hemoglobin in the bloodstream of a human patient. 
It is also an object of the present invention to provide a method of 
treating burn victims. 
It is still another object of the present invention to employ the 
principles of genetic engineering to modify human hemoglobins to improve 
oxygen delivery for the treatment of solid tumors by radiation therapy. 
The above objects and other objects, aims, advantages and goals are 
satisfied by the present invention. 
The present invention concerns a substantially pure polynucleotide coding 
for an alpha and/or beta human globin, the globin when part of a 
hemoglobin molecule confering lower oxygen affinity than normal hemoglobin 
to result in a mutant hemoglobin, the mutant hemoglobin to have an oxygen 
affinity measured for stripped hemoglobin characterized by a P.sub.50 of 
30 torr to 3 atmospheres (2,280 torr) and/or by a Hill coefficient between 
2.5 and 1.0. 
The invention also concerns a replicable recombinant DNA cloning vehicle 
having an insert comprising the aforementioned polynucleotide. The 
invention further relates to a cell that is transfected, infected or 
injected with a recombinant cloning vehicle as described above. 
The invention also concerns a peptide encoded by the aforementioned 
polynucleotide and produced by recombinant DNA technology, a DNA coding 
for such peptide, an expression vector comprising such DNA and a host 
organism transformed with such expression vector. 
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 a substantially pure mutant alpha or beta hemoglobin (e.g., human 
hemoglobin), the hemoglobin having a lower oxygen affinity than normal 
hemoglobin and the mutant hemoglobin having an oxygen affinity measured 
for stripped hemoglobin characterized by a P.sub.50 of 30 torr to 3 
atmospheres (2,280 torr) and/or by a Hill coefficient between 2.5 and 1.0. 
Still further, the present invention is directed to 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 substantially pure mutant alpha or beta hemoglobin, the mutant 
hemoglobin having a lower oxygen affinity than normal hemoglobin and the 
mutant hemoglobin having an oxygen affinity measured for stripped 
hemoglobin characterized by a P.sub.50 of 30 torr to 3 atmospheres (2,280 
torr) and by a Hill coefficient between 2 5 and 1.0. 
The present invention is also directed to a method of treating a warm 
blooded animal, e.g., a human, patient exposed to a burn comprising 
administering to the patient a therapeutically effective amount of a 
substantially pure mutant alpha or beta hemoglobin, the mutant hemoglobin 
having a lower oxygen affinity than normal hemoglobin and the mutant 
hemoglobin having an oxygen affinity measured for stripped hemoglobin 
characterized by a P.sub.50 of 30 torr to 3 atmospheres and/or by a Hill 
coefficient between 2.5 and 1.0. 
______________________________________ 
DEFINITIONS 
Amino Axid 3-letter code 
______________________________________ 
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 
______________________________________ 
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"). 
DNA sequence--A linear array of nucleotides connected one to the other by 
phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. 
Codon--A DNA sequence of three nucleotides (a triplet) which encodes 
through mRNA an amino acid, a translation start signal or a translation 
termination signal. For example, the nucleotide triplets TTA, TTG, CTT, 
CTC, CTA and CTG encode the amino acid leucine ("Leu"), TAG, TAA and TGA 
are translation stop signals and ATG is a translation start signal. 
Reading Frame--The grouping of codons during translation of mRNA into amino 
acid sequences. During translation, the proper reading frame must be 
maintained. For example, the sequence GCTGGTTGTAAG may be translated in 
three reading frames or phases, each of which affords a different amino 
acid sequence 
##STR2## 
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. 
Genome--The entire DNA of a cell or a virus. It includes inter alia the 
structural genes coding for the polypeptides of the cell or virus, as well 
as its operator, promoter and ribosome binding and interaction sequences, 
including sequences such as the Shine-Dalgarno sequences. 
Structural Gene--A DNA sequence which encodes through its template or 
messenger RNA ("mRNA") a sequence of amino acids characteristic of a 
specific polypeptide. 
Transcription--The process of producing mRNA from a structural gene. 
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. When 
the plasmid is placed within a unicellular organism, the characteristics 
of the organism may be changed or transformed as a result of the DNA of 
the plasmid. For example, a plasmid carrying the gene for tetracycline 
resistance (Tet.sup.R) transforms a cell previously sensitive to 
tetracycline into one which is resistant to it. A cell transformed by a 
plasmid is called a "transformant". 
Phage or Bacteriophage--Bacterial virus, many of which consist of DNA 
sequences encapsulated in a protein envelope or coat ("capsid protein"). 
Cloning Vehicle--A plasmid, phage 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. A cloning vehicle is 
often called a vector. 
Cloning--The process of obtaining a population of organisms or DNA 
sequences derived from one such organism or sequence by asexual 
reproduction. 
Recombinant DNA Molecule or Hybrid DNA--A molecule consisting of segments 
of DNA from different genomes which have been joined end-to-end outside of 
living cells and have the capacity to infect some host cell and be 
maintained therein. 
cDNA Expression Vector--A procaryotic cloning vehicle which also contains 
sequences of nucleotides that facilitate expression of cDNA sequences in 
eucaryotic cells. These nucleotides include sequences that function as 
eucaryotic promoters, alternative splice sites and polyadenylation 
signals. 
Transformation/Transfection--DNA or RNA is introduced into cells in such a 
way as to allow gene expression. "Infected" referred to herein concerns 
the introduction of RNA or DNA by a viral vector into the host. 
"Injected" referred to herein concerns the microinjection (use of a small 
syringe) of DNA into a cell. 
Oxygenation Dissociation Curve--Graph showing the relationship of oxygen 
bound to hemoglobin and the ambient partial pressure of oxygen; usually 
expressed as percent saturation of hemoglobin versus the partial pressure 
of oxygen in torr (mm Hg). 
PO.sub.2 --Ambient partial pressure of oxygen usually in torr (1 atmosphere 
equals 760 torr). 
P.sub.50 --The partial pressure of oxygen (PO.sub.2) which corresponds to 
half saturation of the hemoglobin oxygen binding sites. 
n--Hill coefficient (the Hill coefficient for normal hemoglobin is 2.8 to 
3.0). 
Y--Fractional saturation of hemoglobin with oxygen. 
Hill Equation--A mathematical relationship used to parameterize the oxygen 
dissociation curve. The Hill equation is as follows: 
##EQU1## 
After mathematically fitting the Hill equation to the observed oxygen 
dissociation curve the characteristics of the particular curve can be 
described by the values of P.sub.50 and n. 
Bohr Effect--The decrease in hemoglobin oxygen affinity at lower (more 
acidic) pH. 
Root Effect--An extreme Bohr effect observed in some hemoglobins of some 
fish. At acidic pH, circa 6.5, P.sub.50 may reach several atmospheres. 
Stroma-free Hemoglobin--Hemoglobin in solution isolated from the red blood 
cell and red blood cell membrane fragments. 
Stripped Hemoglobin--Hemoglobin in solution free of the inorganic 
phosphates which modulate oxygen affinity (i.e., the 
2,3-diphosphoglycerate normally bound to hemoglobin within the red blood 
cell). Stripped hemoglobin has a pH of generally 10 mmHg. 
Cross-linked Hemoglobin--Hemoglobin modified by covalent bond cross-linkage 
between one or more of the alpha or beta globins. A technique used to 
increase the stability of the hemoglobin alpha-2 beta-2 tetrameric 
structure.

DETAILED DESCRIPTION OF THE INVENTION 
Normal human beta and alpha globin have the following amino acid sequences: 
______________________________________ 
beta alpha 
______________________________________ 
1 Val 
65 Lys 107 Gly 
1 Val 60 Lys 102 Ser 
2 His 
66 Lys 108 Asn 
2 Leu 61 Lys 103 His 
3 Leu 
67 Val 109 Val 
3 Ser 62 Val 104 Cys 
4 Thr 
68 Leu 110 Leu 
4 Pro 63 Ala 105 Leu 
5 Pro 
69 Gly 111 Val 
5 Ala 64 Asp 106 Leu 
6 Glu 
70 Ala 112 Cys 
6 Asp 65 Ala 107 Val 
7 Glu 
71 Phe 113 Val 
7 Lys 66 Leu 108 Thr 
8 Lys 
72 Ser 114 Leu 
8 Thr 67 Thr 109 Leu 
9 Ser 
73 Asp 115 Ala 
9 Asn 68 Asn 110 Ala 
10 Ala 
74 Gly 116 His 
10 Val 69 Ala 111 Ala 
11 Val 
75 Leu 117 His 
11 Lys 70 Val 112 His 
12 Thr 
76 Ala 118 Phe 
12 Ala 71 Ala 113 Leu 
13 Ala 
77 His 119 Gly 
13 Ala 72 His 114 Pro 
14 Leu 
78 Leu 120 Lys 
14 Trp 73 Val 115 Ala 
15 Trp 
79 Asp 121 Glu 
15 Gly 74 Asp 116 Glu 
16 Gly 
80 Asn 122 Phe 
16 Lys 75 Asp 117 Phe 
17 Lys 
81 Leu 123 Thr 
17 Val 76 Met 118 Thr 
18 Val 
82 Lys 124 Pro 
18 Gly 77 Pro 119 Pro 
19 Asn 
83 Gly 125 Pro 
19 Ala 78 Asn 120 Ala 
20 Val 
84 Thr 126 Val 
20 His 79 Ala 121 Val 
21 Asp 
85 Phe 127 Gln 
21 Ala 80 Leu 122 His 
22 Glu 
86 Ala 128 Ala 
22 Gly 81 Ser 123 Ala 
23 Val 
87 Thr 129 Ala 
23 Glu 82 Ala 124 Ser 
24 Gly 
88 Leu 130 Tyr 
24 Tyr 83 Leu 125 Leu 
25 Gly 
89 Ser 131 Gln 
25 Gly 84 Ser 126 Asp 
26 Glu 
90 Glu 132 Lys 
26 Ala 85 Asp 127 Lys 
27 Ala 
91 Leu 133 Val 
27 Glu 86 Leu 128 Phe 
28 Leu 
92 His 134 Val 
28 Ala 87 His 129 Leu 
29 Gly 
93 Cys 135 Ala 
29 Leu 88 Ala 130 Ala 
30 Arg 
94 Asp 136 Gly 
30 Glu 89 His 131 Ser 
31 Leu 
95 Lys 137 Val 
31 Arg 90 Lys 132 Val 
32 Leu 
96 Leu 138 Ala 
32 Met 91 Leu 133 Ser 
33 Val 
97 His 139 Asn 
33 Phe 92 Arg 134 Thr 
34 Val 
98 Val 140 Ala 
34 Leu 93 Val 135 Val 
35 Tyr 
99 Asp 141 Leu 
35 Ser 94 Asp 136 Leu 
36 Pro 
100 Pro 142 Ala 
36 Phe 95 Pro 137 Thr 
37 Trp 
101 Glu 143 His 
37 Pro 96 Val 138 Ser 
38 Thr 
102 Asn 144 Lys 
38 Thr 97 Asn 139 Lys 
39 Gln 
103 Phe 145 Tyr 
39 Thr 98 Phe 140 Tyr 
40 Arg 
104 Arg 146 His 
40 Lys 99 Lys 141 Arg 
41 Phe 
105 Leu 41 Thr 100 Leu 
42 Phe 
106 Leu 42 Tyr 101 Leu 
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 
61 Lys 
62 Ala 
63 His 
64 Gly 
______________________________________ 
The present invention concerns a substantially pure polynucleotide coding 
for a mutant alpha or particularly, beta, human globin. It is preferred to 
make substitutions at human beta positions 90, 102, 108 and combinations 
thereof. 
Preferred specific human beta mutations according to the invention include 
the following: 
(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 lucine.fwdarw.methionine 
amino acid 93 cystine.fwdarw.serine 
amino acid 94 aspartic.fwdarw.glutamic acid (changes amino acid 
86.fwdarw.100 from human beta to trout IV beta) 
The purpose of mutant (6) above is to produce a mutant hemoglobin with a 
Root effect, since it is known that trout hemoglobin IV has a Root effect. 
A preferred alpha mutation would be Asp to Asn at position 94 (hemoglobin 
Titusville). 
The present invention provides the following uses: 
a) Blood replacement stroma-free hemoglobin. Hemoglobin with decreased 
oxygen binding affinity would overcome one of the difficulties associated 
with using stroma-(red cells) free hemoglobin as a blood substitute, i.e., 
an increase in oxygen binding in the absence of red blood cell 2,3-DPG. 
b) 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. 
c) To overcome the problems of hypoxic tumor cells in radiation therapy. 
d) Treatment of normal tissue radiation reactions resulting from vascular 
compromise. 
The present invention serves to deliver more oxygen to tumor cells 
containing hypoxic cells and normal cells containing hypoxic cells due to 
damage by physical or chemical means, e.g., burns, exposure to chemicals, 
physical injuries or ionizing radiation. 
The optimum values for P.sub.50 and the Hill coefficient depend on a number 
of factors. The two are not independent, so that a low value of the Hill 
coefficient would help compensate for a low value of the P.sub.50. The 
following Table recites non-limiting values for three different 
applications, namely, (1) blood replacement, (2) the treatment of vascular 
diseases, i.e., heart attack, stroke, diabetes, normal tissue vascular 
insufficiencies, etc., and for (3) radiation therapy. One set of values is 
given for hemoglobins which do not have a Root effect. For hemoglobins 
which have a Root effect, two sets of values are given, one set for the 
neutral pH at which loading would take place and one set for the acidic pH 
at which unloading would take place. A Root effect hemoglobin would 
probably not be particularly useful for blood replacement. 
TABLE 
__________________________________________________________________________ 
Non-Root Effect 
Root Effect-Mutant Hemoglobins 
Mutant Hemoglobins 
Neutral pH (loading) 
Acid pH (unloading) 
__________________________________________________________________________ 
Blood Replacement 
P.sub.50 
30-50 torr 
n 3.0-2.0 
Vascular Disease 
P.sub.50 
50-350 torr 
P.sub.50 
25-150 torr 
P.sub.50 
&gt;50 torr 
n 3.0-1.0 n 3.0-2.0 
n 3.0-1.0 
Radiation Therapy 
P.sub.50 
75 torr - 3 ATA 
P.sub.50 
25-350 torr 
P.sub.50 
75 torr - 3 ATA 
n 2.5-1.0 n 3.0-1.0 
n 2.5-1.0 
__________________________________________________________________________ 
n = Hill coefficient 
Note: 
Stripped human hemoglobin 
P.sub.50 10 torr 
n = 2.8-3.0 
Human hemoglobin in red blood cell 
P.sub.50 25 torr 
n = 2.8-3.0 
The mutant hemoglobin molecules according to the invention can be 
administered intravenously. They may be formulated in several ways, 
including, as stroma-free hemoglobin, as cross-linked stroma-free 
hemoglobin, contained in natural red blood cells, or contained in 
artificial red blood cells such as liposomes. 
Sufficient hemoglobin would be administered intravenously to provide 
concentrations of from 2 to 12 gm % within the vascular system. For a 
human adult, this would correspond to a total dose of between 100 and 800 
gm hemoglobin. 
However, it may be necessary to deviate from the dosages mentioned and, in 
particular, to do so as a function of the nature and body weight of the 
subject to be treated, the nature and severity of the illness, the nature 
of the preparation and the administration, and the time or interval over 
which the administration takes place. 
Thus, it can suffice in some cases to manage with less than the 
abovementioned amount of hemoglobin while in other cases the 
abovementioned amount of hemoglobin must be exceeded. The particular 
required optimum dosage can easily be decided by anyone skilled in the art 
on the basis of their expert knowledge. 
FIG. 1 depicts the cloning of human beta globin cDNA in E. coli expression 
vectors (M13 and pProk-1). The human beta globin cDNA (Nco1-HindIII) was 
cloned under the control of the tac promoter into two different E. coli 
expression vectors. To facilitate the synthesis of single-stranded DNA for 
oligonucleotide mutagenesis, the beta cDNA was cloned into an M13 
expression vector (mptac 18, Burroughs-Wellcome Laboratories, Langley 
Court, Bahenham, Kent BR3 EB5 England). The beta globin cDNA was also 
cloned into a conventional E. coli expression vector, pProk-1. 
FIG. 2 depicts a technique of oligonucleotide-directed site-specific 
mutagenesis. Mutations in the human .beta. globin gene were made by 
site-directed mutagenesis with specific oligonucleotides. The procedure 
followed was based on that of Kunkel, T.A., Roberts, J.D., Zakour, R., 
(1987), "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic 
Expression", Meth Enzymol. 154:367-382 and Zoller, M.J. and Smith M., 
(1983), "Oligonucleotide-directed Mutagenesis of DNA Fragments Cloned into 
M13 Vectors", Meth Enzymol, 100:468-500. All mutations were sequenced by 
the dideoxy DNA sequencing method. 
FIG. 3 depicts low affinity mutants of human beta globin constructed by 
site-specific mutagenesis. Single amino acid substitutions were made at 
amino acid 90 and amino acid 108. Two different oligonucleotides were 
used; the oligonucleotide for the mutations at amino acid 90 was a mixed 
oligonucleotide. M13 single-stranded DNA was used as the template for the 
mutagenesis. 
FIG. 4 depicts a human trout-IV hybrid beta-globin chain synthesized by 
oligonucleotide-directed mutagenesis using M13 single-stranded DNA as a 
template. Four amino acid substitutions were made by introducing single 
base pair mismatches in the oligonucleotide. This region of the trout beta 
globin protein may impart low oxygen affinity on the human beta chain. 
FIG. 5 depicts low oxygen affinity mutants of human beta globin constructed 
by oligonucleotide insertion mutagenesis. Two amino acid substitutions 
were introduced at amino acid 102 by oligonucleotide insertion mutagenesis 
in a double-stranded expression vector, pProk-1. Two complementary 
oligonucleotides were synthesized in the restriction sites, BamH1 and 
EcoR1 at the ends. These were annealed and this fragment was exchanged for 
the corresponding normal BamH1-EcoR1 fragment of the human beta globin 
gene. 
The invention will now be described with reference to the following 
non-limiting examples. 
EXAMPLES 
Reagents 
Oligonucleotides were synthesized on an Applied Biosystems (Forster City, 
Calif.) DNA Synthesizer model 380B. Restriction enzymes were obtained from 
New England Biolabs (Beverly, Mass.) Sequenase and the Sequenase DNA 
sequencing kit were obtained from United States Biochemicals (Cleveland, 
Ohio). 
Plasmids and E. coli Strains 
The normal human beta globin cDNA was obtained from the University of 
Wisconsin (Madison, Wis.). pPROK-1 was obtained from Clontech (Palo Alto, 
Calif.). mptac18 was obtained from The Wellcome Research Laboratories, 
England. E. coli strain CJ236 was obtained from Yale University, New 
Haven, Conn. 
EXAMPLE 1 
Insertional Mutagenesis of the Beta Globin cDNA 
The normal human beta globin cDNA was cut with BamH1 and EcoR1, which cut 
within the protein coding region and includes AA 102. Two complementary 
oligonucleotides, each 67 bases in length and bearing BamH1 and EcoR1 ends 
were synthesized. The contain a mixture of mutations which changes the 
codon at amino acid (AA) 102 from AAC (Asn) to AGC (Ser, Hb Beth Israel) 
or ACC (Thr, Hb Kans.). The sequence of the oligonucleotides is 
##STR3## 
The oligonucleotides were purified, kinased, annealed together and then 
ligated into the BamH1-EcoR1 cut beta cDNA. The presence of the inserted 
oligonucleotides was confirmed by the addition of a new AvrII site and the 
lack of Bstx1 site when compared to the normal beta cDNA. The inserted DNA 
was sequenced using the dideoxy sequencing method (Sequenase kit) on 
double-stranded plasmid DNA through both cloning sites. 
EXAMPLE 2 
Subcloning into pPROK-1 
The Nco1-HindIII fragment of the mutated beta globin cDNAs, containing the 
complete protein coding region for the beta globin chain, were subcloned 
into the Nco1-HindIII sites of plasmid pPROK-1, and E. coli expression 
vector which uses a tac promoter. 
EXAMPLE 3 
Site-specific mutagenesis of beta globin cDNA 
To facilitate cloning of the Nco1-HindIII fragment of the normal beta 
globin cDNA into mptac18, an Nco1 site was introduced into mptac18 by 
site-specific mutagenesis. mptac18 is an E. coli bacteriophage expression 
vector which uses the tac promoter and produces single-stranded DNA 
suitable for site-specific mutagenesis. A 30-mer which introduces this 
mutation in the polylinker region of mptac18 was synthesized and used in 
the mutagenesis reaction. Mutagenesis was performed using the strains 
developed by Kunkel, supra, and the procedure of Zoller and Smith, supra. 
This is outlined in FIG. 2. Mutants were screened by the presence of a new 
Nco1 site in M13 RF. 
The Nco1-HindIII fragment of the beta globin cDNA was subcloned into the 
mutated mptac18 vector. The presence of the insert was confirmed by 
dideoxy sequencing (Sequenase kit) of the single-stranded DNA. 
Site-directed mutagenesis was performed using the following 
oligonucleotides. 
##STR4## 
This changes the codon at AA 90 from GAG (Glu) to CAG (Gln) and AAG (Lys, 
Hb Agenogi). 
##STR5## 
This changes the codon at AA 108 from AAC (Asn) to GAC (Asp). 
##STR6## 
This changes these codons: 
##STR7## 
Mutagenesis was performed using the E. coli strains developed by Kunkel 
supra and the procedure developed by Zoller and Smith supra. The sequence 
of the mutants was determined by dideoxy DNA sequencing of the 
single-stranded DNA template (Sequenase kit). 
EXAMPLE 4 
Expression of beta globin clones 
Alpha and beta globin clones have been expressed successfully in a number 
of different ways. Simply, they can be expressed in either prokaryotes, 
such as E. coli of eukaryotes, such as mammalian cells. 
Expression in E. coli requires that the gene be cloned behind an E. coli 
transcription promoter, that a Shine-Dalgarno sequence (ribosome binding 
sites) be present in the mRNA and that the protein be stable in order to 
facilitate purification. Alpha and beta globin chains have been expressed 
using a lambda cII promoter (Nagai, K., Perutz, M.F. and Poyart, C., 
(1985), "Oxygen Binding Properties of Human Mutant Hemoglobins Synthesized 
in Escherichia coli.", Proc. Natl. Acad. Sci. USA, 82:7252-7255; Luisi, 
B.F. and Nagai, K., (1986), "Crystallographic Analysis of Mutant Human 
Hemoglobins Made in Escherichia coli.", Nature, 320:555-556). The chains 
were purified and then reconstituted with heme. Other promoters can be 
used for expression including the tac promoter (Brinigar, W.S., Chao, 
T.L., Debouck, C., Gorman, J., Gorman, J.W., Lichenstein, H., O'Donnell, 
J.K., Sutton, J.A. and Young, J.F., (1988), "Expression of Human 
Beta-Globin cDNA in E. coli. Streptomyces and Yeast", Abstract from the 
Symposium on Oxygen Binding Heme Proteins: Structure, Dynamics, Function 
and Genetics, Oct. 9-13, 1988). 
Two of applicants' mutants, Hb Kansas and Hb Beth Israel, are cloned into 
pPROK-1, and E. coli expression vector which uses the tac promoter. 
Expression of the mutant globin chains can be induced by IPTG, the protein 
purified and then re-associated with alpha chain and heme. The other 
mutants described hereinabove are cloned into a single-stranded expression 
vector under the control of a tac promoter (mptac18). This vector has been 
used to express the reverse transcriptase gene from HIV (Larder, B., 
Pinfoy, D., Powell, D. and Darby, G., (1987), "AIDS Virus Reverse 
Transcriptase Defined by High Level Expression in Escherichia coli", EMBO 
J, 6:3133-3137). It is also possible to express both chains 
simultaneously, as has been demonstrated in Hela cells (Stacey, D.W. and 
Allfrey, V.G., (1976), "Microinjection Studies of Duck Globin Messenger 
RNA Translation in Human and Avian Cells", Cell, 9:725-732). This would 
obviate the need for re-association after purification. 
In eukaryotes, globins have been expressed in several different species and 
cell types. Beta globin genes have been successfully expressed in yeast 
(Brinigar, et al, 1988, supra) as well as in several mammalian systems. 
Beta globin genes have been expressed in vivo in hematopoietic stem cells 
of mice (Karlsson, S., Van Doren, K., Schweiger, S.G., Nienhuis, A.N. and 
Gluzman, Y., (1986), "Stable Gene Transfer and Tissue-Specific Expression 
of a Human Globin Gene Using Adenovial Vectors", EMBO J, 5:2377-2385) in 
transgenic mice (Chada, K., Magram, J., Raphael, K. Radice, G., Lacy, E. 
and Costantini, F., (1985), "Specific Expression of a Foreign Beta-Globin 
Gene in Erythroid Cells of Transgenic Mice", Nature. 314:377-380; Soriano, 
P., Cone R.D., Mulligan R.-C. and Jaenisch, R., (1986) "Tissue-Specific 
and Ectopic Expression of Genes Introduced into Transgenic Mice by 
Retroviruses", Science, 234:1409-1413 and Townes, T.M., Lingrel, J.B., 
Chen, H.-Y., Brinster, R.L. and Palmiter, R.D., (1985), 
"Erythroid-Specific Expression of Human Beta-Globin Genes in Transgenic 
Mice, EMBO J, 4:1715-1723) and in vitro in mouse erythroleukemia cells 
(Charnay, P., Treisman, R., Mellon, P., Chao, M., Axel, R. and Maniatis, 
T., (1984), "Differences in Human Alpha and Beta Globin Gene Expression in 
Mouse Erythroleukemia Cells: The Role of Intragenic Sequences", Cell. 
38:251). Again, in these or analogous systems, one or both chains could be 
expressed (Stacey and Allfrey, 1976, supra). 
It will be appreciated that the instant specification and claims are set 
forth by way of illustration and not limitation, and that various 
modifications and changes may be made without departing from the spirit 
and scope of the present invention.