Modified hemoglobin-like compounds and methods of purifying same

The present invention relates to modified hemoglobin-like polypeptides containing multiple dialpha (or dibeta) domains. The present invention also relates to multimeric hemoglobin-like proteins comprising covalently joined hemoglobin-like moieties. Another aspect of the inention is directed at a purification method of hemoglobin-like polypeptides utilizing ion exchange chromatography.

BACKGROUND OF THE INVENTION 
The present invention is directed to modified hemoglobin-like compounds, 
and more particularly to modified hemoglobin-like polypeptides and 
proteins. The present invention is directed also to methods of purifying 
such modified hemoglobin-like compounds. 
Hemoglobin (referred to herein as "Hb") is the oxygen-carrying component of 
blood. Hemoglobin circulates through the bloodstream inside small 
enucleate cells called erythrocytes (red blood cells). Hemoglobin is a 
protein constructed from four associated polypeptide chains, and bearing 
prosthetic groups known as hemes. The erythrocyte helps maintain 
hemoglobin in its reduced, functional form. The heme iron atom is 
susceptible to oxidation, but may be reduced again by one of two enzyme 
systems within the erythrocyte, the cytochrome b.sub.5 and glutathione 
reduction systems. 
Hemoglobin binds oxygen at a respiratory surface (skin, gills, trachea, 
lung, etc.) and transports the oxygen to inner tissues, where it is 
released and used for metabolism. In nature, low molecular weight 
hemoglobins (16-120 kilodaltons) tend to be enclosed in circulating red 
blood cells, while the larger polymeric hemoglobins circulate freely in 
the blood or hemolymph. 
The structure of hemoglobin is well known as described in Bunn & Forget, 
eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders 
Co., Philadelphia, Pa.: 1986) and Fermi & Perutz "Hemoglobin and 
Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in 
Biology (Clarendon Press: 1981). 
About 92% of normal adult human hemolysate is Hb A.sub.o (designated 
alpha.sub.2 beta.sub.2 because it comprises two alpha and two beta 
chains). In a hemoglobin tetramer, each alpha subunit is associated with a 
beta subunit to form a stable alpha/beta dimer, two of which in turn 
associate to form the tetramer. The subunits are noncovalently associated 
through Van der Waals forces, hydrogen bonds and salt bridges. The amino 
add sequences of the alpha and beta globin polypeptide chains of Hb 
A.sub.o are given in Table 1 of PCT Publication No. WO 93/09143. The 
wild-type alpha chain consists of 141 amino acids. The iron atom of the 
heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole 
of His 87 (the "proximal histidine"). The wild-type beta chain is 146 
residues long and heme is bound to it at His 92. 
The human alpha and beta globin genes reside on chromosomes 16 and 11, 
respectively. Bunn and Forget, infra at 172. Both genes have been cloned 
and sequenced, Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha-globin 
genomic DNA); Marotta, et al., J. Biol. Chem., 252:5040-53 (1977) (beta 
globin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomic DNA). 
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of 
the hemoglobin molecule (the two alpha globins and two beta globins in the 
case of Hb A.sub.o), and this cooperativity greatly facilitates efficient 
oxygen transport. Cooperativity, achieved by the so-called heme-heme 
interaction, allows hemoglobin to vary its affinity for oxygen. 
Cooperativity can also be determined using the oxygen dissociation curve 
(described below) and is generally reported as the Hill coefficient, "n" 
or "n.sub.max." Hemoglobin reversibly binds up to four moles of oxygen per 
mole of hemoglobin. 
Oxygen-carrying compounds are frequently compared by means of a device 
known as an oxygen dissociation curve. This curve is obtained when, for a 
given oxygen carrier, oxygen saturation or content is graphed against the 
partial pressure of oxygen. For Hb, the percentage of saturation increases 
with partial pressure according to a sigmoidal relationship. The P.sub.50 
is the partial pressure at which the oxygen-carrying species is half 
saturated with oxygen. It is thus a measure of oxygen-binding affinity; 
the higher the P.sub.50, the more readily oxygen is released. 
The ability of hemoglobin to alter its oxygen affinity under physiological 
conditions, increasing the efficiency of oxygen transport around the body, 
is largely dependent on the presence of the metabolite 
2,3-diphosphoglycerate (2,3-DPG). The oxygen affinity of hemoglobin is 
lowered by the presence of 2,3-DPG. Inside the erythrocyte 2,3-DPG is 
present at a concentration nearly as great as that of hemoglobin itself. 
In the absence of 2,3-DPG "conventional" hemoglobin (hemoglobin A.sub.o) 
binds oxygen very strongly at physiological oxygen partial pressures and 
would release little oxygen to respiring tissue. Accordingly, any 
substitute for hemoglobin must somehow correct the oxygen affinity and/or 
the Hill coefficient to physiologically meaningful levels (see e.g., 
Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, 
L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Hoffman et al., WO 
90/13645; Hoffman and Nagai, U.S. Pat. No. 5,028,588; Anderson et al., WO 
93/09143; Fronticelli, C. et al., U.S. Pat. No. 5,239,061; and De Angelo 
et al., WO 93/08831 and WO 91/16349). 
It is not always practical or safe to transfuse a patient with donated 
blood. In these situations, use of a red blood cell ("RBC") substitute is 
desirable. When human blood is not available or the risk of transfusion is 
too great, plasma expanders can be administered. However, plasma 
expanders, such as colloid and crystalloid solutions, replace only blood 
volume, and not oxygen carrying capacity. In situations where blood is not 
available for transfusion, a red blood cell substitute that can transport 
oxygen in addition to providing volume replacement is desirable. 
To address this need, a number of red blood cell substitutes have been 
developed (Winslow, R. M.(1992) Hemoglobin-based Red Cell Substitutes, The 
Johns Hopkins University Press, Baltimore 242 pp). These substitutes 
include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 
0307087), stroma-free hemoglobin solutions, both chemically crosslinked 
and uncrosslinked, derived from a variety of mammalian red blood cells 
(Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, 
L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Vlahakes, G. J. et al., 
(1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed 
in and purified from genetically engineered organisms (for example, 
non-erythrocyte cells such as bacteria and yeast, Hoffman et al., WO 
90/13645; bacteria, Anderson et al., WO 93/09143, bacteria and yeast 
Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., 
WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 
92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood 
cell substitutes have been designed to replace or augment the volume and 
the oxygen carrying capability of red blood cells. 
However, red blood cell replacement solutions that have been administered 
to animals and humans have exhibited certain adverse events upon 
administration. These adverse reactions have included hypertension, renal 
failure, neurotoxicity, and liver toxicity (Winslow, R. M., (1992) 
Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, 
Baltimore 242 pp.; Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. 
Biotech. 20: 1013-1020). In the case of perfluorocarbons, hypertension, 
activation of the reticulo-endothelial system, and complement activation 
have been observed (Reichelt, H. et al., (1992) in Blood Substitutes and 
Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. supra, pp. 
778-781). For hemoglobin-based oxygen carriers, renal failure and renal 
toxicity are the result of the formation of hemoglobin .alpha./.beta. 
dimers. The formation of dimers can be prevented by chemically 
crosslinking (Sehgal, et al., U.S. Pat. Nos. 4,826,811 and 5,194,590; 
Walder, J. A. U.S. Reissue Pat. RE34271) or genetically linking (Hoffman, 
et al, WO 90/13645) the hemoglobin dimers so that the tetramer is 
prevented from dissociating. 
Prevention of dimer formation has not alleviated all of the adverse events 
associated with hemoglobin administration. Blood pressure changes and 
gastrointestinal effects upon administration of hemoglobin solutions have 
been attributed to vasoconstriction resulting from the binding of 
endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et 
al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., 
Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992) 
Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. 
Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived 
relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et 
al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and 
constitutive NO are primarily produced in the endothelium of the 
vasculature and act as local modulators of vascular tone. 
When hemoglobin is contained in red blood cells, it cannot move beyond the 
boundaries of blood vessels. Therefore, nitric oxide must diffuse to the 
hemoglobin in an RBC before it is bound. When hemoglobin is not contained 
within an RBC, such as is the case with hemoglobin-based blood 
substitutes, it may pass beyond the endothelium lining the blood vessels 
and penetrate to the extravascular space (extravasation). Thus, a possible 
mechanism causing adverse events associated with administration of 
extracellular hemoglobin may be excessive inactivation of nitric oxide due 
to hemoglobin extravasation. Furthermore, NO is constitutively synthesized 
by the vascular endothelium. Inactivation of NO in the endothelium and 
extravascular space may lead to vasoconstriction and the pressor response 
observed after infusions of cell-free hemoglobin. Larger hemoglobins may 
serve to reduce hypertension associated with the use of some extracellular 
hemoglobin solutions. 
In addition to the effects noted above, the dosage of non-polymeric 
extracellular hemoglobin that can be administered may be limited by the 
colloidal osmotic pressure (COP) of the solution. Administration of an 
extracellular hemoglobin composed of hemoglobin tetramers that would have 
the same grams of hemoglobin as a unit of packed red blood cells might 
result in a significant influx of water from the cells into the blood 
stream due to the high colloid osmotic pressure of the hemoglobin 
solution. Polymeric hemoglobin solutions can be administered at higher 
effective hemoglobin dosages, because as the molecular weight increases, 
the number of the individual molecules is decreased, resulting in reduced 
COP (Winslow, R. M., (1992) Hemoglobin-based Red Cell Substitutes, The 
Johns Hopkins University Press, Baltimore, pp 34-35). 
Some higher molecular weight hemoglobins occur in nature. For example, 
there are three mutants of human hemoglobin that are known to polymerize 
as a result of formation of intermolecular (first tetramer to second 
tetramer) disulfide bridges. Tondo, Biochem. Biophys. Acta, 342:15-20 
(1974) and Tondo, An. Acad. Bras. Cr., 59:243-251 (1987) describe one such 
mutant known as Hb Porto Alegre. Hb Mississippi is characterized by a 
cysteine substitution in place of Ser CD3(44).beta. and is believed to be 
composed of ten or more hemoglobin tetramers according to Adams et al., 
Hemoglobin, 11(5):435-542 (1987). Hemoglobin Ta Li is characterized by a 
.beta.83(EF7)Gly.fwdarw.Cys mutation, which showed slow mobility in starch 
gel electrophoresis, indicating that it too was a polymer. 
There are a few known naturally occurring mutants of non-polymerizing human 
hemoglobins that have a cysteine mutation that do not polymerize (Harris 
et al., Blood, 55(1):131-137 (1980)(Hemoglobin Nigeria); Greer et al., 
Nature [New Biology], 230:261-264 (1971) (Hemoglobin Rainier). Hemoglobin 
Nunobiki (.alpha. 141 Arg.fwdarw.Cys) also features a non-polymerizing 
cysteine substitution. In both Hb Rainier and Hb Nunobiki, the mutant 
cysteine residues are surface cysteines. 
Polymeric hemoglobins have also been reported in various vertebrates and 
invertebrates. Murine polymeric hemoglobins are described in Bonaventura & 
Riggs (Science, (1967)149:800-802) and Riggs (Science, (1965)147:621-623). 
A polymerizing hemoglobin variant in macaque monkeys is reported in 
Takenaka et al., Biochem Biophys. Acta, 492:433-444 (1977); Ishimoto et 
al., J. Anthrop. Soc. Nippon, 83(3):233-243 (1975). Both amphibians and 
reptiles also possess polymerizing hemoglobins (Tam et al., J. Biol. 
Chem., (1986) 261:8290-94). 
Some invertebrate hemoglobins are also large multi-subunit proteins. The 
extracellular hemoglobin of the earthworm (Lumbricus terrestris) has 
twelve subunits, each of which is a dimer of structure (abcd).sub.2 where 
"a", "b", "c", and "d" denote the major heme containing chains. The "a", 
"b", and "c" chains form a disulfide-linked trimer. The whole molecule is 
composed of 192 heme-containing chains and 12 non-heme chains, and has a 
molecular weight of 3800 kDa. The brine shrimp Artemia produces three 
polymeric hemoglobins with nine genetically fused globin subunits 
(Manning, et al., Nature, (1990) 348:653). These are formed by variable 
association of two different subunit types, a and b. Of the eight 
intersubunit linkers, six are 12 residues long, one is 11 residues and one 
is 14 residues. 
Non-polymerizing crosslinked hemoglobins have been artificially produced. 
For example, hemoglobin has been altered by chemically crosslinking the 
alpha chains between the Lys99 of alpha.sub.1 and the Lys99 of alpha.sub.2 
(Walder, U.S. Pat. Nos. 4,600,531 and 4,598,064; Snyder, et al., PNAS 
(USA) (1987) 84: 7280-84; Chatterjee, et al., J. Biol. Chem., (1986) 261: 
9927-37). The beta chains have also been chemically crosslinked 
(Kavanaugh, et al., Biochemistry, (1988) 27: 1804-8). U.S. Pat. No. 
5,028,588 suggests that the T state of hemoglobin (corresponding to 
deoxygenated hemoglobin) may be stabilized by intersubunit (but 
intratetrameric) disulfide crosslinks resulting from substitution of 
cysteine residues for other residues. 
Hemoglobin has also been artificially crosslinked to form polymers. For 
example, U.S. Pat. No. 4,001,401, U.S. Pat. No. 4,001,200, U.S. Pat. No. 
4,777,244 and U.S. Pat. No. 4,053,590 all relate to polymerization of red 
blood cell-derived hemoglobin by chemical crosslinking. The crosslinking 
is achieved with the aid of bifunctional or polyfunctional crosslinking 
agents, especially those reactive with exposed amino groups of the globin 
chains. Aldehydes such as glutaraldehyde and glycolaldehyde have been used 
to crosslink hemoglobin both intramolecularly (within a tetramer) and 
intermolecularly (between tetramers). Intramolecular crosslinks serve to 
prevent dimerization into alpha/beta dimers and may also alter oxygen 
affinity, while intermolecular crosslinks create polymers of tetrameric 
hemoglobin. Polymeric hemoglobins may result in reduced extravasation 
because of their increased size. Reduced extravasation may, in turn, lead 
to reduced pressor effects resulting from infused hemoglobin solutions. 
The result of these polymerization chemistries that have been used to 
crosslink hemoglobins is a polydisperse composition of covalently 
crosslinked aggregates. Bucci, U.S. Pat. No. 4,584,130, at col. 2, 
comments that "the polyhemoglobin reaction products are a heterogeneous 
mixture of various molecular species which differ in size and shape. The 
molecular weights of these polyhemoglobins range from 64,500 to 600,000 
Daltons. The separation of individual molecular species from the 
heterogeneous mixture is virtually impossible. In addition, although 
longer retention times in vivo are obtained using polyhemoglobins, the 
oxygen affinity thereof is higher than that of stroma-free hemoglobin." 
It is well recognized that random polymerization is difficult to control 
and that a number of different polymers can be obtained, commonly between 
two and ten tetramers per polymer. For example, according to Tye, U.S. 
Pat. No. 4,529,179, polymerized pyridoxylated hemoglobin has "a profound 
chemical heterogeneity making it difficult to study as a pharmaceutical 
agent." 
Furthermore, once hemoglobin is polymerized, purification of specific 
molecular weight fractions can be accomplished using only molecular weight 
separation techniques. For example, tangential flow separation techniques 
can be used to separate certain size ranges of polymerized hemoglobins. 
However the membranes that are available for such separations are 
available only in a limited number of size ranges which allow the 
production of hemoglobins less than 100 kDa or greater than 300 kDa. In 
addition, such membranes are cumbersome, expensive, difficult to clean and 
the separation can be very slow. 
Size exclusion chromatography (also known as, for example, gel filtration 
chromatography or gel permeation chromatography) has also been used in the 
past to separate hemoglobin molecular weight fractions. However, this 
technique is not suitable for large scale operation, and furthermore, does 
not provide good resolution for separation of molecular weight fractions 
(Simoni et al., (1993) Anal. Chim. Acta, 279: 73-88). 
Simoni (1993, infra) also report the use of ion exchange chromatography to 
separate different molecular weight fractions of hemoglobin polymers. 
However, these workers noted that this kind of separation required 
differences in net charges. In addition, they used a salt gradient elution 
to separate the different molecular weight fractions, and they did not 
demonstrate any significant resolution of tetramer, octamer and decamer. 
Correlations of molecular weight with serum half life for various proteins, 
such as IL-2, demonstrate that a significantly longer half life may be 
expected as the molecular weight of a protein increases, particularly 
above the renal filtration limit of 50-70 kDa. The use of crosslinkers 
that can inhibit the degradation of hemoglobin tetramers into dimers that 
are readily cleared can also lead to increased serum half life. 
Accordingly, a need exists for additional hemoglobin-like compounds having 
these desired characteristics. In addition, a need exists for simple 
methods of creating specific molecular weight distributions in high 
molecular weight hemoglobin mixtures. The present invention satisfies 
these needs and provides related advantages. 
SUMMARY OF THE INVENTION 
The present invention relates to modified hemoglobin-like compounds. In one 
aspect, the invention is directed to globin-like polypeptides having 
multiple dialpha domains. Such polypeptides can contain two dialpha 
domains, also referred to herein as "di-dialpha" domains, or more. These 
globin-like polypeptides can be linked by a peptide linker having at least 
five amino acids between the dialpha domains, preferably at least seven 
amino adds. Preferably, the linkers are encoded by a peptide linker having 
Ser-Gly-Gly as a repeat unit, such as the amino acid sequences: 
Ser-Gly-Gly-Ser-Gly-Gly-Ser (SEQ.ID.NO.1); 
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (SEQ.ID.NO.2) and 
Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser 
(SEQ.ID.NO.3). The globin-like polypeptides can be recombinantly expressed 
in a host cell, such as E. coli. Di-dibeta globin-like polypeptides are 
analogously defined, and are a further aspect of the instant invention. 
The invention also relates to nucleic add molecules having a nucleic add 
sequence encoding such globin-like polypeptides. In one embodiment, the 
nucleic acid molecules encode a globin-like polypeptide having two dialpha 
domains and a separate polypeptide having a single beta domain or a 
di-beta domain. 
In another aspect, the present invention relates to a multimeric hemoglobin 
compound that comprises two dialpha globins that are connected through a 
peptide linker, wherein only one of the four alpha globin domains contains 
a non-naturally occurring cysteine residue (mono-cys di-dialpha). In a 
further aspect of this invention, such mono-cys di-dialpha-containing 
hemoglobin composition can be crosslinked directly or indirectly to 
another identical mono-cys di-dialpha or any other suitable 
hemoglobin-like molecule. Mono-cys di-dibeta molecules are analogously 
defined and also can be crosslinked as described herein. 
In another aspect, the present invention also provides multimeric 
hemoglobin-like proteins in which a first hemoglobin-like moiety is 
directly attached to two or more other hemoglobin-like moieties. 
Compositions containing such multimeric hemoglobin-like proteins are also 
provided. 
In a further aspect, the present invention relates to methods for making 
the multimeric hemoglobin-like proteins. The methods are accomplished by: 
(a) obtaining a first hemoglobin-like moiety having amino acids capable of 
attaching to one end of a heterobifunctional linker to form a core 
hemoglobin-like moiety; 
(b) obtaining at least two other hemoglobin-like moieties having an amino 
acid capable of attaching to the other end of the heterobifunctional 
linker; 
(c) contacting the heterobifunctional linker to the first hemoglobin-like 
moiety; and 
(d) adding the other hemoglobin-like moieties to form the multimeric 
hemoglobin-like protein. 
In a still further aspect, the present invention relates to methods for 
separation of molecular weight fractions of polymerized hemoglobin or 
hemoglobin-like molecules to obtain substantially monodisperse hemoglobin 
solutions. Such methods are accomplished by: 
(a) contacting a polydisperse mixture of polymerized hemoglobin-like 
molecules with an ion exchange matrix; 
(b) washing the ion exchange matrix with a first buffer; 
(c) eluting the ion exchange matrix with a second buffer which may be the 
same or different than said first buffer to obtain a substantially 
monodisperse hemoglobin-like solution. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention generally relates to hemoglobin-like compounds 
comprised of novel globin-like polypeptides or hemoglobin-like proteins. 
These compounds contain various modifications to the naturally-occurring 
hemoglobins, particularly human Hb A.sub.o. In a further aspect, the 
present invention relates to methods of purifying such hemoglobin-like 
molecules and other polymeric hemoglobin-like molecules. 
As described above, most naturally-occurring human hemoglobins are 
constructed of four non-covalently linked polypeptide chains: two chains 
containing identical alpha domains and two chains containing identical 
beta domains. The novel globin-like polypeptides of the present invention, 
however, contain at least two dialpha (or two dibeta) domains in a single 
polypeptide chain. A "dialpha domain" (or "dibeta domain") consists of two 
alpha (or beta) domains (or polypeptide sequences) connected between the 
C-terminus of a first alpha domain (or beta domain) and the N-terminus of 
a second alpha domain (or beta domain) as described in PCT Publication No. 
WO 93/09143, incorporated herein by reference. Thus, the novel globin-like 
polypeptides have as a minimum four alpha (or beta) domains per 
polypeptide. 
As used herein, the term "globin-like polypeptide" means a polypeptide 
having a domain that is substantially homologous with a globin subunit of 
a naturally occurring hemoglobin. For example, a globin-like polypeptide 
containing two dialpha domains means that each of the four alpha domains 
is substantially homologous to a native alpha globin or a mutant thereof 
differing from the native sequence by one or more substitutions, deletions 
or insertions, while remaining substantially homologous with the native 
alpha globin and retaining its ability to associate with a beta globin. As 
used herein, the term "alpha domain" is intended to include but not be 
limited to naturally occurring alpha globins, including normal human alpha 
globin, and mutants thereof. A "beta domain" is analogously defined. 
Subunits of vertebrate and invertebrate hemoglobins or mutants thereof 
which are sufficiently homologous with human alpha or beta globin are 
embraced by the terms "alpha or beta domains." For example, the subunits 
of bovine hemoglobin are within the scope of these terms. 
In determining whether an alpha or beta globin contemplated by the present 
invention is substantially homologous to a particular wild-type alpha or 
beta globin, sequence similarity is an important but not exclusive 
criterion. Sequence similarity may be determined by conventional 
algorithms, which typically allow introduction of a small number of gaps 
in order to achieve the best fit. An alpha domain contemplated for use in 
the present invention will typically have at least about 75% sequence 
identity with wild-type human alpha globin, and greater homology with 
human alpha globin than with human beta globin. However, a polypeptide 
having an alpha domain of lesser sequence identity may still be considered 
"substantially homologous" with a wild-type alpha globin if it has a 
greater sequence identity than would be expected from chance and also has 
the characteristic higher structure (e.g., the "myoglobin fold") of alpha 
globin. 
Mutations can be introduced to alter the oxygen affinity (or cooperativity, 
or activity with respect to pH, salt, temperature, or other environmental 
parameters) or stability (to heat, acid, alkali, or other denaturing 
agents) of the hemoglobin, to facilitate genetic fusion or crosslinking, 
or to increase the ease of expression and assembly of the individual 
chains. Guidance as to certain types of mutations is provided, for 
example, in U.S. Pat. No. 5,028,588 and PCT Publication No. WO 93/09143, 
both incorporated herein by reference. The present invention further 
includes molecules which depart from those taught herein by gratuitous 
mutations that do not substantially affect biological activity. 
The dialpha (or dibeta) domains of the novel globin-like polypeptides can 
be connected by various means known in the art. For example, the domains 
can be coupled by a peptide linker between any two dialpha domains. A 
discussion of suitable distances is also provided in WO 93/09143, 
incorporated herein by reference. With knowledge of these distances, one 
skilled in the art can readily determine, for example through molecular 
modeling, the useful lengths of suitable peptide linkers. Particularly 
useful peptide linkers have at least five amino acids, preferably at least 
seven amino acids. The peptide linker can have an amino acid sequence that 
contains Ser-Gly-Gly as a repeating unit, as in the following illustrative 
amino add sequence: Ser-Gly-Gly-Ser-Gly-Gly-Ser (SEQ.ID.NO. 1). Examples 
of other amino acid sequences useful as peptide linkers containing this 
repeating unit include: 
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (SEQ.ID.No. 2) and 
Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser 
(SEQ.ID.No. 3). 
The multiple dialpha domains and the peptide linkers of the globin-like 
polypeptides can be genetically fused through recombinant methods known in 
the art or as described, for example, in WO 93/09143 or in the Examples 
below. The preparation of a single dialpha globin as an intermediate 
product is also described in this publication. 
The globin-like polypeptides can be used to prepare hemoglobin-like 
pseudomers. Such pseudomeric Hb-like proteins are described in WO 
93/09143. Pseudomeric hemoglobin-like proteins have at least one more 
domain than the number of polypeptide chains, i.e., at least one 
polypeptide chain contains two or more globin-like domains. 
It is also possible to introduce non-naturally occurring cysteine residues 
into one alpha subunit of a dialpha domain or one alpha subunit of a 
di-dialpha domain or larger dialpha domains to prepare other pseudomeric 
hemoglobin-like proteins. Preferably these non-naturally occurring 
cysteine residues are asymmetric, that is they occur in only one alpha 
domain of the longer di-dialpha polypeptide. Such mutations can also be 
incorporated in an analogous fashion in di-dibeta globins. The asymmetric 
cysteine residues can then be used to form direct disulfide bridges 
connecting the dialpha (or dibeta) domains or crosslinked by coupling 
reagents specific for cysteine residues to produce the larger pseudomeric 
Hb proteins. 
The hemoglobin-like pseudomers can be purified by any suitable purification 
method known to those skilled in the art. Useful purification methods for 
the hemoglobin-like proteins of the present invention are taught in PCT 
Publication WO 95/14038, incorporated herein by reference. Briefly, the 
methods described therein involve an immobilized metal affinity 
chromatography resin charged with a divalent metal ion such as zinc, 
followed by anion exchange chromatography. According to this publication, 
the solution containing the desired Hb-containing material to be purified 
can first be heat treated to remove protoporphyrin IX-containing Hb. This 
basic purification method can be further followed by a sizing column 
(S-200), then another anion exchange column. Alternatively, this solution 
can be separated into molecular weight fractions using ion exchange 
chromatography according to the methods of the instant invention. The 
resulting solution can then be buffer exchanged to the desired formulation 
buffer. 
The invention further provides nucleic adds encoding the novel polypeptides 
of the present invention. Those skilled in the art can readily derive a 
desired nucleotide sequence based on the knowledge of published nucleotide 
or amino add sequences of known hemoglobin subunits with selection of 
codons and control elements specific for the organism used for expression, 
using methods known in the art. For example, the amino add sequence of the 
dialpha domain and the beta domain of a synthetic hemoglobin can be used 
to derive the nucleic acids of the present invention, both of which are 
identified in FIG. 12 of PCT Publication WO 90/13645, incorporated herein 
by reference, with the following corrections to the nucleotide sequence: 
bases 55, 56 and 57 (codon 19) should read GCG and bases 208 and 209 (the 
first two bases of codon 70) should read GC. The following changes to the 
amino add sequence of this figure would yield the pseudotetramer, rHb1.1: 
the gly-gly bridge at residues 142 and 143 of the dialpha domain can be 
changed to a single gly residue bridging .alpha..sub.1 and .alpha..sub.2 
domains; residues 54 and 97 of the dialpha domain should read Gln; residue 
70 of the beta subunit should read Asn; and residue 107 of the beta 
subunit should read Lys. The pseudotetramer, rHb1.1 is also described in 
Looker et al., Nature, 356:258-260 (1992), incorporated herein by 
reference. 
The nucleic acids of the present invention can be used to construct 
plasmids to be inserted into appropriate recombinant host cells according 
to conventional methods or as described in the Examples below. Any 
suitable host cell can be used to express the novel polypeptides. Suitable 
host cells include, for example, bacterial, yeast, mammalian and insect 
cells. E. coli cells are particularly useful for expressing the novel 
polypeptides. Preferably, when multiple subunits are expressed in 
bacteria, it is desirable, but not required, that the subunits be 
co-expressed in the same cell polycistronically as described in WO 
93/09143. The use of a single promoter is preferable in E. coli to drive 
the expression of the genes encoding the desired proteins. 
The present invention is also directed to novel multimeric hemoglobin-like 
proteins containing at least three hemoglobin-like moieties, of which at 
least one is directly attached to the other moieties. The term 
"hemoglobin-like moiety" includes tetramers having four globin-like 
domains composed of two alpha domains and two beta domains and pseudomeric 
hemoglobin-like proteins as previously defined. The hemoglobin-like moiety 
that is directly attached to the other hemoglobin-like moieties is 
referred to herein as the "core hemoglobin-like moiety" or "core moiety" 
while the other hemoglobin-like moieties are referred to as the 
surrounding hemoglobin-like moieties" or "surrounding moieties." 
In one embodiment, the core moiety is different from the surrounding 
hemoglobin-like moieties, which in turn can be the same or different from 
each other. Such multimeric hemoglobin-like proteins are referred to as 
heteromultimeric hemoglobin-like proteins (or heteromers). For example, 
the core moiety can be rHb1.1, while the surrounding moieties can be 
mutants referred to as K158C. The pseudotetramer, rHb1.1, is described in 
WO 90/13645, incorporated herein by reference. K158C is a mutant moiety of 
rHb1.1 and is composed of three polypeptides, one containing two alpha 
domains (a dialpha) and the other two each containing a single beta 
domain. A single lysine to cysteine substitution in the second alpha 
domain of the dialpha component appears at amino acid residue 158 of the 
K158C dialpha sequence. Note that because rHb1.1 consists of a dialpha 
molecule (two alpha subunits, each 141 amino acids in length, connected by 
a single glycine) mutations in the second subunit are denoted by the 
position with respect to the N terminus of the dialpha, and not the alpha 
subunit. Thus the mutation at position 158 is a mutation in the second 
alpha globin domain, corresponding to position 16 in normal alpha globin. 
A general method for obtaining a moiety having one or more asymmetrical 
cysteine mutations and the desirability of such asymmetrical crosslinked 
mutants are provided in WO 93/09143, which is specifically incorporated 
herein by reference. The publication also provides guidance for selecting 
other candidate sites for substitution on the alpha or beta domains. 
The core and surrounding moieties can be directly attached by any means 
known in the art, including without limitation the use of chemical 
crosslinkers. Such linkers are discussed in Wang, S. S. (1993) Chemistry 
of Protein Conjugation and Crosslinking, CRC Press. Other suitable 
crosslinking methods are described, for example in Vandegriff, K. D.(1992) 
Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. 
Tombs, Editor, Intercept Ltd., Andover, England; and Winslow, R. M. (1992) 
Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, 
Baltimore 242 pp. Such crosslinking chemistries are generally linkers 
containing two or more functional groups. These functional groups can be 
the same or different (i.e., homobifunctional linkers, heterobiftunctional 
linkers, homopolyfunctional linkers, or heteropolyfunctional linkers and 
can furthermore be dendrimeric, branched or contain armed cores) and 
include, for example, bis-imidoesters, bis-succinimidyl esters, oxidized 
ring structures of sugars or nucleotides, crosslinkers containing 
haloacetyl or vinyl sulfone functional groups, and dialdehyde and 
polyaldehyde crosslinkers, such as glycolaldehyde and glutaraldehyde. 
For heteromultimeric hemoglobin-like proteins, a heterobifunctional 
chemical crosslinker is preferred, such as a succinimidyl 
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or 
N-.gamma.-maleimidobutyrloxysuccinimide ester (GMBS). Preferably, the 
heterobifunctional chemical crosslinker is one that does not elicit a 
significant immunogenic response. Other useful heterobifunctional 
crosslinkers are described in WO 93/09143, incorporated herein by 
reference. 
In tire case of GMBS or SMCC, for example, the succinimide of these 
compounds can be used to attach to the lysine residue of the non-cysteine 
mutated hemoglobin-like proteins, such as rHb1.1 (the core moiety). The 
maleimide can be used to attach to the cysteine of the hemoglobin-like 
protein containing a cysteine mutation, such as K158C. By first reacting 
linkers with the core moiety, then adding the desired amount of 
cysteine-containing mutant, various forms of these multimeric 
hemoglobin-like proteins can be made, for example a trimeric, tetrameric, 
pentameric and higher order multimeric proteins. Factors that constrain 
the number of hemoglobin-like moieties that can be attached to the core 
moiety include steric hindrance as additional surrounding moieties are 
added and the number of residues that are available for attaching to the 
crosslinkers. Methods for identifying and using such crosslinkers are 
known to those skilled in the art or as described in the Examples below. 
In a further embodiment, the core moiety and the surrounding moieties can 
be the same moiety, which are referred to herein as "homomultimeric 
hemoglobin-like proteins." An example of a homomultimeric Hb-like protein 
is one which is composed of only K158C mutants. 
For making the multimeric hemoglobin-like proteins of this embodiment, the 
formation of substantial amounts of polymerized proteins is preferably 
avoided. Polymerized proteins contain Hb-like moieties that are indirectly 
attached to the core moiety through attachment to an intervening 
hemoglobin-like moiety and are generally formed by uncontrolled 
crosslinking reactions. According to the methods of the instant invention, 
such random polymerization is reduced by coupling of specific reactive 
sites on the core hemoglobin protein to certain sites on the surrounding 
hemoglobin-like molecules. Any method known in the art can be used in 
which site specific attachment can be achieved. 
The present invention also provides methods for making homomultimeric 
hemoglobin-like proteins, that is a multimeric hemoglobin-like protein 
composed of a core molecule that is the same as the surrounding molecules. 
These methods are accomplished by the use of a heterobifunctional 
crosslinker and a protective moiety, for example, borate. Alternatively, 
reaction conditions with any of the crosslinkers can be modified by 
altering for example, concentrations, temperatures or reaction time such 
that the degree of polymerization is constrained. 
Through the use of an appropriate amine/sulfhydryl heterobifunctional 
crosslinker, a desired hemoglobin-like moiety, for example, rHb1.1, can be 
modified so that it will subsequently react with several rHb1.1 molecules 
bearing surface cysteine mutations as described in WO 93/09143. This 
reaction is achieved, for example, by first reacting the amine 
functionalities on unmodified rHb1.1 with the succinimide moiety of a 
heterobifunctional crosslinker in a sodium borate buffer at pH 8.5. 
Reaction with lysine residues on rHb1.1 leads to loss of the succinimide 
group of the heterobifunctional crosslinker by the formation of a stable 
amide linkage between the crosslinker and the hemoglobin. The unreacted 
maleimide residues of the heterobifunctional crosslinker are highly 
reactive towards sulfhydryl groups. The intrinsic sulfhydryl groups of 
rHb1.1 are prevented from reacting with the maleimide moiety of the 
heterobifunctional crosslinker by either their inaccessibility or by 
forming a complex with borate. After reaction with the succinimide group 
of the heterobifunctional crosslinker, the hemoglobin molecule can be 
considered to be "activated" at multiple surface lysine residues towards 
reaction with the surface sulfhydryl residue of, for example, a K158C 
hemoglobin mutant because the core moiety now has reactive maleimide 
residues attached to it. 
By using an appropriate concentration of crosslinker and reaction time, 
which can be determined empirically by those skilled in the art, the 
reaction with surface cysteine-containing hemoglobin (e.g., K158C) with 
the activated core hemoglobin molecule yields higher molecular weight 
hemoglobins. The polymers that are formed by reaction of the activated 
hemoglobin and the cysteine-containing hemoglobin mutants have a 
distribution of apparent molecular weights. However, the distribution of 
molecular weights can be constrained to a certain extent by the extent of 
initial activation with the heterobifunctional crosslinker coupled with 
the use of certain moieties, such as, for example, K158C. The 
site-directed nature of the reaction with, for example, K158C limits the 
molecular weight distribution to predominantly pentameric hemoglobin. It 
is believed that the manipulation of reactivity, such as sulfhydryl 
reactivity, through formation of a reversible complex with a suitable 
protective buffer, such as borate/boric acid for certain mutants, is a 
novel method for controlling reactivity, such as sulfhydryl reactivity, in 
forming the multimeric hemoglobin-like proteins of the present invention. 
Accordingly, the present invention further provides methods for making a 
multimeric hemoglobin-like protein. The methods are accomplished by: 
(a) obtaining a first hemoglobin-like moiety having an amino acid capable 
of attaching to one end of a heterobifunctional linker to form a core 
hemoglobin-like moiety; 
(b) obtaining at least two other hemoglobin-like moieties having an amino 
acid capable of attaching to the other end of the heterobifunctional 
linker; 
(c) contacting the heterobifunctional linker to the first hemoglobin-like 
moiety to form a linked moiety; and 
(d) contacting the other hemoglobin-like moieties to the linked moiety to 
form the multimeric hemoglobin-like protein. 
The invention further provides compositions containing the novel multimeric 
hemoglobin-like proteins of the present invention and the globin-like 
polypeptides, including proteins containing such polypeptides. In 
compositions containing the multimeric hemoglobin-like proteins, a 
polydisperse composition containing various multimeric proteins can be 
obtained, i.e., differing species of trimerics, tetramerics, pentamerics 
and so forth. In addition, these compositions containing the multimeric 
hemoglobin-like proteins are preferably substantially free of polymerized 
proteins, although they need not be completely free depending on the 
intended use of the desired proteins. As used in this context, 
"substantially free" means the presence of polymerized proteins will not 
adversely affect the desired function of the multimeric hemoglobin-like 
proteins. Furthermore, these multimeric hemoglobin-like proteins are 
substantially monodisperse. As used herein, "substantially monodisperse" 
means that there is less than 30% hemoglobin that is not the desired 
molecular weight. Accordingly, in a substantially monodisperse high 
molecular weight hemoglobin solution, less than 30% of the hemoglobin is 
not the target high molecular weight hemoglobin that is desired. Note that 
the target monodisperse high molecular weight hemoglobin can comprise 
mixtures of high molecular weight, such as trimers, tetramers and 
pentamers. Likewise, in a substantially monodisperse pentahemoglobin 
solution, less than 30% of the hemoglobin in the solution is not 
pentahemoglobin. Preferably, a monodisperse high molecular weight 
hemoglobin solution contains less than 25% non-target hemoglobin, more 
preferably less than 20% non-target hemoglobin. 
After crosslinking, regardless of crosslinking technology that is utilized, 
ion exchange chromatography is used to separate hemoglobin polymers by 
molecular weight according to the methods of the instant invention. 
Typically ion exchange chromatography is used to separate proteins 
according to differences in isoelectric point. Surprisingly, the inventors 
have found that ion exchange technology can be used to separate 
hemoglobins that have no measurable difference in isoelectric point. For 
example, the isoelectric point of rHb1.1 that has been crosslinked with 
glutaraldehyde is approximately 7.05 for mono-hemoglobin (1 tetramer) 
di-hemoglobin (2 tetramers), tri-hemoglobin, and higher order multimers. 
Nevertheless, such hemoglobins were resolved using the methods of the 
instant invention (see, for example, Example 12 herein). 
According to the instant invention, the purification of the polydisperse 
hemoglobin solution is accomplished as follows. The polydisperse 
hemoglobin solution is transferred into a buffer compatible with the ion 
exchange matrix if required. A suitable buffer is, for example, 20 mM 
Tris, pH 8.0-8.9 at 8.degree. C. The polydisperse hemoglobin solution is 
then loaded onto an ion exchange matrix. Such ion exchange matrices can be 
any suitable support, for example membranes or resins that are anion or 
cation exchange matrices. A particularly suitable exchange resin can be, 
for example, a Q-SEPHAROSE fast flow anion-exchange column (Pharmacia 
Biotech, Uppsala, Sweden). Alternate anion-exchange resins include, for 
example, Super Q 650 C or Toyopearl QAE-550C (TosoHaas Inc., Montgomery, 
Pa.) or Macro-Prep Q Support (Bio-Rad Inc., Hercules, Calif.). The amount 
of protein that can be loaded on the column can be varied depending on the 
binding capacity of the column and the mix of molecular weights desired. 
The flow rate of the column will depend on the type of column and resin 
used for the chromatography. Typically, for a 450 ml resin bed packed in 
an XK-50 column (Pharmacia Biotech, Uppsala, Sweden) a flow rate of 200 
cm/hr is used. 
After the column is loaded with the polydisperse hemoglobin solution, it is 
washed with sufficient column volumes of buffer to remove unbound protein 
from the column matrix. Such washes can be, for example, 2-3 column 
volumes (CV's) of 20 mM Tris buffer, pH 8.9 at 8.degree. C. Alternatively, 
the column can be washed until the desired protein concentration in the 
eluent is reached. This can be determined by the absorbance at 215 or 280 
nm, or by other suitable monitoring techniques. Next, the column is washed 
with the desired buffer system. This buffer system can include 
combinations of buffer, buffer concentrations and/or salt to elute the 
desired protein. Elution can occur utilizing any suitable elution scheme, 
for example by isocratic elution, stepwise isocratic elution, stepwise 
gradient elution or gradient elution. A particularly suitable elution 
scheme is by stepwise isocratic elution. Determination of suitable washes, 
elution buffers and elution schemes can be readily determined by one of 
skill in the art using the guidance provided herein. 
For the purpose of removing glutaraldehyde crosslinked proteins with 
molecular weights &lt;190 kDa the column can be washed with, for example, 11 
CV's of 20 mM Tris buffer, pH 7.6 at 8.degree. C. Using this system, the 
desired hemoglobin polymer fraction is then eluted with 20 mM Tris, pH 7.4 
at 8.degree. C. Likewise, monomeric hemoglobin can be removed from a 
pentahemoglobin solution formed using a polyfunctional crosslinker (see 
Example 18) using, for example, 7-8 CV wash 25 mM Bis-Tris/Tris pH=7.5 at 
8.degree. C. followed by elution with 25 mM Bis-Tris/Tris, 100 mM NaCl 
pH=7.5 at 8.degree. C. The hemoglobin molecular weight fraction of 
interest can then formulated as desired, or further purified by, for 
example, ultrafiltration. 
The hemoglobin-like proteins and compositions containing the globin-like 
polypeptides or the multimeric hemoglobin-like proteins (collectively 
"hemoglobins") can be used for in vitro or in vivo applications. Such in 
vitro applications include, for example, the delivery of oxygen by 
compositions of the instant invention for the enhancement of cell growth 
in cell culture by maintaining oxygen levels in vitro (DiSorbo and Reeves, 
PCT publication WO 94/22482, herein incorporated by reference). Moreover, 
the hemoglobins of the instant invention can be used to remove oxygen from 
solutions requiring the removal of oxygen (Bonaventura and Bonaventura, 
U.S. Pat. No. 4,343,715, incorporated herein by reference) and as 
reference standards for analytical assays and instrumentation (Chiang, 
U.S. Pat. No. 5,320,965, incorporated herein by reference) and other such 
in vitro applications known to those of skill in the art. 
In a further embodiment, the hemoglobins of the present invention can be 
formulated for use in therapeutic applications. Example formulations 
suitable for the hemoglobin of the instant invention are described in 
Milne, et al., WO 95/14038 and Gerber et al., PCT/US95/10232, both herein 
incorporated by reference. Pharmaceutical compositions of the invention 
can be administered by, for example, subcutaneous, intravenous, or 
intramuscular injection, topical or oral administration, large volume 
parenteral solutions useful as blood substitutes, etc. Pharmaceutical 
compositions of the invention can be administered by any conventional 
means such as by oral or aerosol administration, by transdermal or mucus 
membrane adsorption, or by injection. 
For example, the hemoglobins of the present invention can be used in 
compositions useful as substitutes for red blood cells in any application 
that red blood cells are used or for any application in which oxygen 
delivery is desired. Such hemoglobins of the instant invention formulated 
as red blood cell substitutes can be used for the treatment of 
hemorrhages, traumas and surgeries where blood volume is lost and either 
fluid volume or oxygen carrying capacity or both must be replaced. 
Moreover, because the hemoglobins of the instant invention can be made 
pharmaceutically acceptable, the hemoglobins of the instant invention can 
be used not only as blood substitutes that deliver oxygen but also as 
simple volume expanders that provide oncotic pressure due to the presence 
of the large hemoglobin protein molecule. In a further embodiment, the 
crosslinked hemoglobin of the instant invention can be used in situations 
where it is desirable to limit the extravasation or reduce the colloid 
osmotic pressure of the hemoglobin-based blood substitute. The hemoglobins 
of the present invention can be synthesized with a high molecular weight. 
Thus the hemoglobins of the instant invention can act to transport oxygen 
as a red blood cell substitute, while reducing the adverse effects that 
can be associated with excessive extravasation. 
A typical dose of the hemoglobins of the instant invention as an oxygen 
delivery agent can be from 2 mg to 5 grams or more of extracellular 
hemoglobin per kilogram of patient body weight. Thus, a typical dose for a 
human patient might be from a few grams to over 350 grams. It will be 
appreciated that the unit content of active ingredients contained in an 
individual dose of each dosage form need not in itself constitute an 
effective amount since the necessary effective amount could be reached by 
administration of a plurality of administrations as injections, etc. The 
selection of dosage depends upon the dosage form utilized, the condition 
being treated, and the particular purpose to be achieved according to the 
determination of the skilled artisan in the field. 
Administration of the hemoglobins of the instant invention can occur for a 
period of seconds to hours depending on the purpose of the hemoglobin 
usage. For example, as an oxygen delivery vehicle, the usual time course 
of administration is as rapid as possible; Typical infusion rates for 
hemoglobin solutions as blood replacements can be from about 100 ml to 
3000 ml/hour. 
In a futher embodiment, the hemoglobins of the instant invention can be 
used to treat anemia, both by providing additional oxygen carrying 
capacity in a patient that is suffering from anemia, and/or by stimulating 
hematopoiesis as described in PCT publication WO 95/24213. When used to 
stimulate hematopoiesis, administration rates can be slow because the 
dosage of hemoglobin is much smaller than dosages that can be required to 
treat hemorrhage. Therefore the hemoglobins of the instant invention can 
be used for applications requiring administration to a patient of high 
volumes of hemoglobin as well as in situations where only a small volume 
of the hemoglobin of the instant invention is administered. 
Because the distribution in the vasculature of the hemoglobins of the 
instant invention is not limited by the size of the red blood cells, the 
hemoglobin of the present invention can be used to deliver oxygen to areas 
that red blood cells cannot penetrate. These areas can include any tissue 
areas that are located downstream of obstructions to red blood cell flow, 
such as areas downstream of thrombi, sickle cell occlusions, arterial 
occlusions, angioplasty balloons, surgical instrumentation, any tissues 
that are suffering from oxygen starvation or are hypoxic, and the like. 
Additionally, any types of tissue ischemia can be treated using the 
hemoglobins of the instant invention. Such tissue ischemias include, for 
example, stroke, emerging stroke, transient ischemic attacks, myocardial 
stunning and hibernation, acute or unstable angina, emerging angina, 
infarct, and the like. Because of the broad distribution in the body, the 
hemoglobins of the instant invention can also be used to deliver drugs and 
for in vivo imaging. 
The hemoglobins of the instant invention can also be used as replacement 
for blood that is removed during surgical procedures where the patient's 
blood is removed and saved for reinfusion at the end of surgery or during 
recovery (acute normovolemic hemodilution or hemoaugmentation). In 
addition, the hemoglobins of the instant invention can be used to increase 
the amount of blood that can be predonated prior to surgery, by acting to 
replace some of the oxygen carrying capacity that is donated. 
Under normal physiological conditions, nitric oxide is not produced in 
excess amounts. However, certain disease states are associated with excess 
nitric oxide production. Such conditions include septic shock and 
hypotension. In these cases, the crosslinked hemoglobin of the present 
invention can be used to remove excess nitric oxide from the vasculature 
or to remove any other ligand that is found in toxic excess and that can 
be bound to the hemoglobins of the instant invention. 
The following Examples are intended to illustrate, but not limit, the 
present invention.

EXAMPLE 1 
Production of Protein Solution Containing Modified Hemoglobin 
A. Construction of a Bacterial System for the Production of Modified rHb1.1 
On Jan. 20, 1994 E. coli strain SGE1661 was deposited with the American 
Type Culture Collection (ATCC Accession Number 55545). Note that Strain 
SGE1661 carrying the plasmid pSGE705 was denoted SGE1662. pSGE705 was a 
medium copy number plasmid because it resulted in approximately 100 copies 
of the plasmid per cell. The plasmids used in preparing pSGE705 are 
identified in Table 1, which also provides a brief description of each. 
Materials. pBR322, pUC19 and pNEB193 were purchased from New England 
Biolabs, Beverly, Mass. Oligonudeotides were synthesized on an Applied 
Biosystems DNA Synthesizer Model 392. The oligonudeotides used in 
preparing pSGE705 are listed in Table 2. Restriction endonucleases were 
purchased from New England Biolabs (Beverly, Mass.) and used according to 
manufacturer's specifications. T4 DNA Ligase was purchased from either New 
England Biolabs or Gibco-BRL (Gaithersburg, Md.) and used according to 
manufacturer's specifications. Pfu polymerase was purchased from 
Stratagene (La Jolla, Calif.) and used according to manufacturer's 
specifications. 
Media used to culture the strains are described in J. H. Miler, Experiments 
in Molecular Genetics. (Cold Spring Harbor Press 1972). and J. H. Miller, 
A Short Course in Bacterial Genetics. (Cold Spring Harbor Press 1992). 
Acridine orange, ampicillin and kanamycin sulfate were purchased from 
Sigma Chemical Co. (St Louis, Mo.). Tetracycline was purchased from 
Aldrich Chemicals (Milwaukee, Wis.). 
TABLE 1 
______________________________________ 
Plasmids 
PLASMID DESCRIPTION 
______________________________________ 
pSGE1.1E4 
rHb1.1 expression plasmid containing dialpha and 
beta genes 
pSGE1.1E5 like pSGE1.1E4 but ampicillin resistant instead of 
tetracycline resistant 
pSGE490 pUC19 lacI on a Bam HI-Hind III fragment 
pSGE491 pUC19 .alpha. on an Eco RI-Xba I fragment 
pSGE492 pNEB193 Ptac- .alpha. 
pSGE493 pUC19 .beta. on an Xba I-Hind III fragment 
pSGE500 pUC19 .alpha. .beta. on a Bam HI-Hind III fragment 
pSGE504 pSELECT-1 replace Sty I with a Pme I site 
pSGE505 pSGE504 rrnB T1 transcriptional terminator in the Eco 
RI-Cla I sites 
pSGE507 ColE1 ori and tet, 2213 bp 
PSGE509 ColE1 ori tet lacI, 3425 bp 
pSGE513 ColE1 ori tet lacI .alpha. .beta., 4386 bp 
pSGE515 ColE1 ori tet lacI di.alpha. .beta. , 4812bp 
pSGE700 pTZ18U + di.alpha. .beta. from pSGE515 
pSGE705 modified rHb1.1 expression plasmid, ColE1 ori, tet, lacI, 
dialpha and beta genes 
pTZ18U a phagemid derivative of pUC19, for oligonucleotide 
directed mutagenesis 
pDLII-91F pGEM1 + .alpha. missing valine in 2nd position (Des-val) 
pNEB193 Like pUC19 but has more restriction sites in the multi 
cloning sites 
pBRr322 ColE1 ori tet amp 
pRG1 pACYC177 lacIq 
______________________________________ 
TABLE 2 
__________________________________________________________________________ 
Oligonucleotides 
OLIGO SEQUENCE (5'-3') DESCRIPTION 
__________________________________________________________________________ 
EV18 CGGGAATACGGTCTAGATCATTAA 
C-term of .alpha. gene, 
SEQ. ID #4 CGGTATTTCGAAGTCAGAACG Xba I site 
EV27 GATCCGAGCTGTTGACAATTAATCATCGGCT tac promoter 
SEQ. ID #5 CGTATAATGTGTGGAATTGTGACGGATAACAA 
sequence, Bam HI- 
TTTCACACAGGAAATTAATTAATGCTGTCTCC Eag I sites 
EV28 GGCCGGAGACAGCATTAATTAATTTCCTGT tac promoter 
SEQ. D #6 GTGAAATTGTTATCCGCTCACAATTCCACA 
sequence, Bam HI- 
CATTATACGAGCCGATGATTAATTGTCAAC Eag I sites, 
AGCTCG complement of EV27 
EV29 TCGGATTCGAATTCCAAGCTGTTGGATCC 
TTA 5' end of .alpha. with Eco RI, 
SEQ. ID #7 GATTCAACTGTCTCCGGCCGATAAAACCACCG 
Bam HI andEag I sites 
EV30 CGGAAGCCCAATCTAGAGGAAATAATATAT 5' end of .beta. 
with 
SEQ. ID #8 GCACCTGACTCCGGAAGAAAAATCC Xba I site 
EV31 CCCGAAACCAAGCTTCATTAGTGA 3' end of the .beta. 
gene 
SEQ. ID #9 GCTAGCGCGTTAGCAACACC with Hind III site 
MW007 TTTAAGCTTCATTAGTGGTATT 
mutagenesis reverse primer 
SEQ. ID #10 TGTGAGCTAGCGCGT replaces last 3 codons 
of .beta. 
missing in pSGES15 
MW008 CAGCATTAATTAACCTCCTTA mutagenesis reverse 
SEQ. ID #11 GTGAAATTGTTATCCG 
primer to optimize .alpha. 
ribozyme binding site (RBS) 
MW009 GGTGCATATATTTACCTCCTT 
mutagenesis reverse primer 
SEQ. ID #12 ATCTAGATCATTAACGGTATTTCG to optitize .beta. RBS; 
remove 
2nd Bg1 II 
TG14 GGTTTAAACC Pme I linker 
SEQ. ID #13 
TG59 GGCGAATAAAAGCTTGCGGCCGCG Upstream of lacI 
gene, has 
SEQ. ID #14 TTGACACCATCGAATGGCGCAAAA Hind III and Not I site 
CCTTTCGCGG- 
upstream of promoter 
TG60 GGGCAAATAGGATCCAAAAAAAAG Downstream side of 
lacI 
SEQ. ID #15 CCCCCTCATTAGGCGGGCTTTAT gene with trp transcripti 
onal 
CACTGCCCGCTTTCCAGTCGGG terminator and Bam HI 
site 
TG62 CCCCGAAAAGGATCGAAGTA upstream primer for 
pBR322 
SEQ. ID #16 GCCCGCGGCCGCGTTCCACTG ori positions 3170-3148 
AGCGTCAGACCCC 
w/Bam HI and Not I site 
TG63 GGCGGTCCTGTITAAACGCT downstream primer for 
SEQ. ID #17 GCGCTCGGTCGTTCGGCTGCGG pBR322 ori positions 
2380-2404 w/Pme I site 
__________________________________________________________________________ 
Genetic and Molecular Biological Procedures. Standard bacterial genetic 
procedures are described in J. H. Miller, Experiments in Molecular 
Genetics, (Cold Spring Harbor Press 1972) and J. H. Miller, A Short Course 
in Bacterial Genetics (Cold Spring Harbor Press, 1992 ). Standard 
molecular biology procedures were performed as described in Sambrook et 
al., Molecular Cloning, (Cold Spring Harbor Press, 1989). 
Plasmid DNA Transformation. DNA transformations were performed by the 
procedure described in Wensick et al., Cell 3: 315-325 (1974). Briefly, 
cells were grown to mid log phase and then pelleted, resuspended in an 
equal volume of 10 mM MgSO.sub.4 and incubated on ice for 30 minutes. The 
cells were centrifuged and the pellet resuspended in 1/2 original volume 
of 50 mM CaCl.sub.2 and placed on ice for 20 minutes. The cells were 
centrifuged again and then resuspended in 1/10 original volume of 50 mM 
CaCl.sub.2. Plasmid DNA was added to the competent cells in a solution of 
10 mM Tris-HCl pH 8.0, 10 mM MgCl.sub.2 and 10 mM CaCl.sub.2. The mixture 
was incubated on ice for 15 minutes and then incubated at 37.degree. C. 
for 5 minutes. One milliliter of LB medium was added and the mixture 
incubated with shading for 30-60 minutes. The culture was then 
centrifuged, resuspended in 0.1 ml of LB medium and plated on the 
appropriate selective medium. 
Purification of DNA. DNA fragments were purified from an agarose gel using 
the Geneclean system (Bio 101, Inc. La Jolla, Calif.) according to the 
method provided with product. PCR products were prepared and cleaved with 
restriction endonucleases using the Double Geneclean system. (Bio 101, 
Inc. La Jolla; method provided with product.) Briefly, the PCR product was 
purified away from the PCR primers, then the PCR product was cleaved with 
restriction endonuclease(s) and purified from the restriction endonuclease 
and buffer. The PCR product was then ready for a ligation reaction. 
Annealing of oligonudeotides. Complementary oligonudeotides were annealed 
according to the following procedure. Equimolar amounts of each 
oligonucleotide were mixed in 15-25 .mu.l of 10 mM Tris-HCl pH 8.0/1 mM 
EDTA and incubated at 65.degree. C. for 30 minutes. The sample was 
transferred to a 37.degree. C. water bath for 30 minutes. Finally, the 
sample was incubated on ice for 60 minutes or in the refrigerator 
overnight. 
Oligonucleotide directed mutagenesis. Oligonucleotide directed mutagenesis 
was performed with the Muta-gene phagemid in vitro mutagenesis kit 
(Bio-Rad, Hercules, Calif.) according to manufacturer's instructions which 
are based on the method of Kunkel (Kunkel, T. A. (1985) Proc. Natl. Acad. 
Sci. USA 82: 488; Kunkel et al., (1987) Methods Enzymol. 154: 367). The 
rHb1.1 region of pSGE515 was cloned into pTZ18U (Bio-Rad, Hercules, Calif. 
or U.S. Biochemical, Cleveland, Ohio) on a BamHI-HindIII fragment to 
create pSGE700. Three oligonudeotides, MW007, MW008 and MW009 were used to 
simultaneously introduce multiple changes in a single reaction. 
Preparation of pBR322 ori. PCR primers were designed to amplify the pBR322 
origin of replication. These primers, TG62 and TG63, annealed to the 
positions 2380-2404 and 3170-3148 on the pBR322 DNA sequence (Sutcliffe, 
J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43: 77-90). The PCR 
product was digested with NotI and PmeI. The DNA fragment was purified 
according to the Geneclean procedure. 
Preparation of tet gene fragment. The source for the tet gene was pSELECT-1 
(Promega Corp., Madison, Wis.). This plasmid has a number of restriction 
endonuclease sites, such as BamHI, HindIII, SalI and SphI removed from the 
tet gene (Lewis and Thompson (1993) Nucleic Adds Res. 18:3439-3443). A 
PmeI linker was inserted into the StyI site of pSELECT-1. This plasmid was 
designated pSGE504. Oligonudeotides TG71 and TG72 were annealed and 
ligated to the EcoRI-ClaI fragment of pSGE504. This plasmid, pSGE505, was 
shown to have the expected restriction endonuclease sites and to have lost 
the sites present in the multicloning site of pSELECT-1. pSGE505 was 
digested with NotI and PmeI. The 1417 bp fragment was purified according 
to the Geneclean protocol. 
Preparation of lacI gene. The lacI gene was isolated by amplifying the gene 
sequence from pRG1 (Dana-Farber Cancer Inst, Boston) that carried the lacI 
gene. The PCR primers, TG59 and TG60 were designed to generate a wild type 
lacI promoter (Farabaugh, P. J. (1978) Nature 274:765), upstream of the 
gene and to place the trp terminator sequence (Christie et al., (1981) 
Proc. Natl. Acad. Sci. USA 78:4180-4184) downstream of the gene. The same 
step could be carried out using Y1089 (Promega) or chromosomal DNA from 
any E. coli strain carrying the lac region, such as MM294 (ATCC 33625.) 
The PCR product was gel purified and isolated according to the Geneclean 
procedure and cloned into BamHI-HindIII digested pUC19 DNA to make 
pSGE490. 
Construction of pSGE515. PCR primers EV29 and EV18 were chosen to amplify 
the alpha gene from pDLII-91F (Hoffman et al., WO 90/13645). The purified 
PCR product was cleaved with the restriction endonucleases EagI and XbaI. 
To create a plasmid that contained P.sub.tac -.alpha., the alpha gene (from 
above) and the tac promoter, which was prepared by annealing EV27 and 
EV28, were mixed with Eco RI-Xba I-cleaved pUC19 DNA. The mixture of the 
three DNA fragments, in approximately equimolar ratio, was treated with T4 
DNA Ligase. After incubation the ligation mixture was used to transform 
SGE476 and ampicillin resistant transformants were selected. 
(Transformation into Strain MM294 (ATCC 33625) yields equivalent results.) 
An isolate with the correct restriction endonuclease fragments was 
designated pSGE492. The a gene and the tac promoter DNA sequences were 
verified by DNA sequencing. 
Primers EV30 and EV31 were used to amplify the .beta. gene from pSGE1.1E4 
by PCR. The purified .beta. gene fragment was digested with XbaI and 
HindIII and then mixed with XbaI-HindIII digested pUC19 DNA and treated 
with T4 DNA ligase. The ligation mixture was used to transform competent 
SGE476 (equivalent to MM294, ATCC 33625) and transformants were selected 
on LB+ampicillin (100 .mu.g/ml) plates. An isolate that contained the 
appropriate restriction endonuclease fragments was chosen and designated 
pSGE493. The .beta. gene was confirmed by DNA sequencing. 
The .beta. gene was isolated from pSGE493 by restriction with XbaI and 
HindIII followed by purification according to the Geneclean method. This 
DNA fragment was then ligated to XbaI-HindIII restricted pSGE492 DNA and 
transformed into SGE713. (Any dam.sup.- strain such as JM110 (ATCC 47013) 
or GM119 (ATCC 53339) could also be used.) An ampicillin resistant 
transformant that carried a plasmid that had the appropriate restriction 
fragments was chosen and designated pUC19.alpha..beta. (pSGE500). 
The BamHI-Hind III fragment that contained the .alpha. and .beta. genes of 
pSGE500 was purified according to the Geneclean method. An XhoI fragment 
that carried a portion of the di-.alpha. gene containing the glycine 
linker region was gel purified from pSGE1.1E5. pSGE1.1E5 (described in 
Hoffman et al., U.S. Ser. No. 789,179, filed Nov. 8, 1991) is a 
tetracycline sensitive analogue of pSGE1.1E4 (Hoffman et al., WO 
90/13645), which could also have been used. 
The pBR322 origin of replication region (pBR322 ori, above) was ligated to 
the tet gene fragment (above) and the ligation mixture was transformed 
into SGE476. (Transformation into MM294, above would yield equivalent 
results.) Tetracycline resistant transformants were selected and plasmid 
DNA was isolated and analyzed. An isolate that contained the appropriate 
restriction endonuclease fragments was chosen and designated pSGE507. 
Next, pSGE507 and pSGE490 were digested with BamHI and NotI and the 
appropriate fragments were purified. The two purified fragments were 
ligated together and the ligation mixture was used to transform competent 
SGE713. (Any dam.sup.- strain could also be used; see above.) Tetracycline 
resistant transformants were selected, and plasmid DNA was isolated and 
analyzed. A plasmid that had the appropriate restriction fragments was 
chosen and designated pSGE509. 
The purified BamHI-HindIII fragment of pSGE500 that contained the .alpha. 
and .beta. genes was ligated to BamHI-HindIII digested pSGE509. The 
ligation mixture was used to transform pSGE713 (see above for equivalent 
strains) and tetracycline resistant transformants were selected and 
characterized. An isolate yielding the correct size plasmid with the 
expected restriction endonuclease fragments was chosen and designated 
pSGE513. 
The XhoI fragment of pSGE1.1E (described in Hoffman et al., U.S. Ser. No. 
07/789,179, filed Nov. 8, 1991, now U.S. Pat. No. 5,545,727, incorporated 
herein by reference) that contained the di-.alpha. glycine linker sequence 
was ligated to XhoI digested pSGE513 to create a plasmid that contained 
the di-.alpha. gene. SGE753 was transformed with the ligation mixture and 
tetracycline resistant transformants were selected. (Transformation into 
SGE800 would have yielded equivalent results.) Isolates were screened to 
identify those that contained the XhoI fragment inserter into pSGE513 in 
the correct orientation. An isolate that contained the correct 
configuration of the di-.alpha. gene, as determined by restriction 
endonuclease analysis with EagI, was designated pSGE515. 
Modification of pSGE515 to create pSGE705. The DNA sequence record used to 
design PCR primers for the amplification of the .beta. gene did not 
contain the C-terminal three amino acids. Oligonucleotide directed 
mutagenesis was used to add these nine nucleotides to the DNA sequence of 
the .beta. gene. In the same reactions, modifications were introduced to 
optimize the ribosome binding sites for the di-.alpha. and .beta. genes, 
and to remove a BglII site near the end of the di-.alpha. gene. The 
HindIII-BamHI fragment from pSGE515 was subcloned into pTZ18U, creating 
pSGE700. pSGE700 was then used as a source of ssDNA for site-directed 
mutagenesis. 
The following are the changes that were made with the oligonucleotides 
MW008 and MW009 to optimize ribosomal binding sites and to remove a BglI 
restriction endonuclease site. 
di alpha 
before - CAATTTCAC--AGGAAATTAATTAATGCTG (SEQ. ID. NO. 25) 
.vertline..vertline..vertline..vertline..vertline..vertline..ve 
rtline..vertline..vertline.**.vertl 
ine..vertline..vertline..vertline.* 
*.vertline..vertline..vertline..ver 
tline..vertline..vertline..vertline 
..vertline..vertline..vertline..ver 
tline..vertline..vertline. 
after - CAATTTCACTAAGGAGGTTAATTAA 
TGCTG (SEQ. ID. NO. 26) 
Four nucleotide changes, shown above, including the insertion of two 
nucleotides, were introduced with MW008 to optimize the ribosome binding 
site for dialpha. (.vertline.-indicates identity, *-indicates a change) 
beta 
before - TAAaGATCTAGA---GGAAATAA-TATATGCAC (SEQ. ID. NO. 27) 
.vertline..vertline..vertline.*.vertline..vertline..vertline..v 
ertline..vertline..vertline..vertl 
ine..vertline.***.vertline..vertli 
ne..vertline.**.vertline..vertline 
..vertline.*.vertline..vertline..v 
ertline..vertline..vertline..vertl 
ine..vertline..vertline..vertline. 
after - TAATGATCTAGATAAGGAGGTAAATATATGCAC (SEQ. ID. NO. 28) 
The six nucleotide changes shown above, including the insertion of four 
nucleotides, were introduced with MW009 to optimize the ribosome binding 
site for beta. The lower case "a" on the before strand was a T to A 
mutation in the construction of the alpha gene that introduced a Bgl II 
site into the sequence. This was removed so that there would only be a 
single Bgl II site in pSGE705. (.vertline.-indicates identity, *-indicates 
a change) 
End of Beta 
before - CTCGCTCAC---------TAATGAA (SEQ.ID.NO.29) 
.vertline..vertline..vertline..vertline..vertline..vertline..ve 
rtline..vertline..vertline.*********.vertline..vert 
line..vertline..vertline..vertline..vertline..vertl 
ine. 
after - CTCGCTCACAAATACCACTAATGAA (SEQ.ID.NO.30) 
MW007 introduced the coding sequence for the last three amino adds of the 
beta gene as shown above. (.vertline.-indicates identity, *-indicates a 
change) 
Putative mutants were screened for loss of a BglII restriction endonuclease 
cleavage site (introduced by MW008). Seventeen of 24 had lost the site and 
were further characterized by DNA sequencing at the other two mutagenized 
sites. One of the 17 had incorporated all the modifications from the three 
oligonucleotides. These changes were verified by DNA sequencing and the 
rHb1.1 genes were cloned into BamHI-HindIII digested pSGE509. An isolate 
that had the correct restriction endonuclease fragments was designated 
pSGE705. 
A new sequence upstream of the a gene minimized the distance between the 
tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21-25, 1983) 
and the first codon of the alpha gene. The intergenic region between the 
di-.alpha. gene and the .beta. gene was also designed to contain the 
minimum sequence that contained a restriction endonuclease site and the 
ribosome binding site for the .beta. gene. A plasmid map of pSGE705 is 
shown in FIG. 1. The plasmid map indicates many of the restriction 
endonuclease cleavage sites. pSGE705 is smaller than its counterpart 
pSGE1.1E4, and the placement of its restriction sites facilitates modular 
alterations of the sequence. An unused antibiotic resistance marker was 
removed, and a promoter was added to the lacI gene that would allow 
tighter control of rHb1.1. expression. pSGE705 was the base plasmid used 
in all manipulations described in the Examples set forth below. 
General Fermentation Protocol 
Hemoglobin was expressed in the strains described herein using any one of 
the fermentation protocols described below. First, a fermentor inoculum 
was grown from seed stock. An optional 2 liter flask fermentation was then 
performed prior to transfer to a 15 liter fermentor and induction. 
Alternatively, 100 liter fermentations were used. If the latter approach 
was used, then a fermentor inoculum was grown from seed stock 2 liter 
shake flasks. Four of these shake flasks were then used to inoculate the 
100 liter fermentors. The details of the fermentation process are 
described below. Any suitable fermentation and pre-purification scheme 
(purification prior to the ion exchange molecular weight separation) can 
be used for the production of the material of the instant invention. 
Seed Stock-All Fermentations 
Seed stock was grown up in LB broth containing 10 g/L BactoTrypton.TM., 5 
g/L yeast extract, 5 g/L NaCl, 0.2 g/L NaOH, and 10 ug/ml tetracycline to 
an optical density of 1.5-1.7 at 600 nm. The solution was then made up to 
10% glycerol and stored at -80.degree. C. until required. 
15 Liter Fermentation Protocol 
Fermentor Inoculum (500 ml broth in 2 L shake flasks-seed flasks) 
To prepare the fermentor inoculum, seed stock was thawed and 0.1-0.4 ml of 
seed stock were inoculated into 500 ml of a solution (DM-1) containing 
approximately 4.1 g/L KH.sub.2 PO.sub.4, 7 g/L K.sub.2 HPO.sub.4, 2 g/L 
(NH.sub.4).sub.2 SO.sub.4, 1 g/L Na.sub.3 citrate-2H.sub.2 O, 153 mg/L 
MgSO.sub.4.7H.sub.2 O, 2.3 g/L of L-proline, 2 g/L yeast extract, 4.8-5.5 
g/L glucose, 320 mg/L thiamine HCl, 10 mg/L tetracycline, and 3 ml/L of a 
trace metal solution containing 32.5 mg/L FeCl.sub.3.6H.sub.2 O, 1.6 mg/L 
ZnCl.sub.2, 2.4 mg/L CoCl.sub.2.6H.sub.2 O, 2.4 mg/L Na.sub.2 
MoO.sub.4.2H.sub.2 O, 1.2 mg/L CaCl.sub.2.2H.sub.2 O, 1.5 mg/L 
Cu(II)SO.sub.4.5H.sub.2 O, 0.06 mg/L H.sub.3 BO.sub.3, and 120.2 ml/L HCl. 
This culture is allowed to grow for 8-10 hours at 37.degree. C. on a 
shaker. Two flasks were combined and used to inoculate the 15L fermentors 
if no intermediate "2 Liter" fermentation was performed. Alternatively, an 
intermediate seed fermentation in two liter fermentors was performed prior 
to the 15 liter fermentation. 
Fermentor (2 L volume-seed fermentation) As 
an optional intermediate step, the cells were grown in a 2 liter 
fermentation. 400 mL of the seed fermentation was then aseptically 
transferred to a 2-liter New Brunswick fermentor containing approximately 
1700 mL of a solution containing approximately: 2.2 g/L KH.sub.2 PO.sub.4, 
4 g/L K.sub.2 HPO.sub.4 and 2.2 g/L (NH.sub.4).sub.2 SO.sub.4. 
The medium in the fermenter also contained: 1.2 g/L trisodium citrate, 1.2 
g/L MgSO.sub.4.7H.sub.2 O, 2.5 g/L proline, 3.1 g/L of the trace metal 
solution described above, 0.1 mg/L tetracycline in 50% ethanol solution, 
345 mg/ L thiamine HCl in purified water, sterile filtered solution, 200 
g/L of 70% glucose, 50+10 g/L of 30% NH.sub.4 OH, and 2 ml PPG 2000 
(polyethylene glycol 2000). 
Cells were grown in the fermentor for approximately 10 hours. The pH was 
maintained at 6.8-6.95 by addition of 15% to 30% NH.sub.4 OH, dissolved 
oxygen was maintained at or above 20%, and 50-70% glucose was added 
throughout the growth period, sufficient to maintain low but adequate 
levels of glucose in the culture (2 g/L-10 g/L). The culture was grown at 
approximately 30.degree. C. to an OD.sub.600 .about.2-5. 
15L Fermentor (14 L volume in 20 L Fermentor-"15L") 
Either 800 mls of the seed flask or 400 mls of the "2 liter" seed fermentor 
were then aseptically transferred to a 20-liter fermentor containing 8 
liters of the following media (DM-4-RP): 1.3 g/L KH.sub.2 PO.sub.4, 2.4 
g/L K.sub.2 HPO.sub.4, 1.3 g/L (NH.sub.4).sub.2 SO.sub.4, 195 mg/L 
thiamine HCl, 6.1 mg/L tetracycline, 1.8 g/L proline, and 2.2 ml/L of the 
trace metal solution described above. Note that masses of added reagents 
are calculated using the final volume of fermentation (11.5 liters) and 
are approximate within measurement error. The pH was maintained at 6.8 to 
6.95 by addition of 15% to 30% NH.sub.4 OH, dissolved oxygen was 
maintained at or above 20%, and 50 to 70% glucose was added throughout the 
growth period, sufficient to maintain low but adequate levels of glucose 
in the culture (2 g/L-10 g/L). Dissolved oxygen was maintained as dose to 
20% as possible. The culture was grown between 28 and 32.degree. C. until 
an OD.sub.600 of 30 was reached. Induction was accomplished by the 
addition of 10-1000 .mu.M isopropyl thiogalactoside (IPTG). Upon induction 
of hemoglobin synthesis, the E. coli heme biosynthesis was supplemented by 
addition of hemin dissolved in 1 N NaOH, either by addition at induction 
of the total mass of hemin required, by continuous addition of hemin 
throughout the induction period, or by periodic addition of hemin 
dissolved in 50 mM to 1 M NaOH (e.g. one third of the total mass of hemin 
to be added to the fermentor was added at induction, another third was 
added after 1/4 of the total time after fermentation had elapsed, and the 
last third was added half-way through the induction period). Total hemin 
added ranged from 50 to 300 mg/L. The fermentor was allowed to continue 
for 8-12 hours post-induction. 
100 Liter Fermentation Protocol 
Fermentor Inoculum (500 mL broth in 2 L shake flasks) 
To prepare the fermentor inoculum, seed stock was thawed. Seed stock (100 
ml) was grown up in 500 ml of DM59 in an Erlenmeyer flask at 37.degree. C. 
in a 1 inch rotary shaker (275 to 300 rpm) for 8 to 10 hours. DM59 media 
is: 3.34 g/L KH.sub.2 PO.sub.4, 5.99 g/L K.sub.2 HPO.sub.4, 1.36 g/L 
NaH.sub.2 PO.sub.4.H.sub.2 O, 1.95 g/L Na2HPO.sub.4, and 1.85 g/L 
(NH.sub.4)SO.sub.4 which are sterilized. After sterilization, 12.20 ml/L 
of a trace metal solution was added. The trace metal solution contained: 
134.2 g/L tripotassium citrate, 32.2 g/L trisodium citrate, 27 g/L 
FeCl.sub.3.6H.sub.2 O, 2.2 g/L ZnCl.sub.2, 0.3 g/L CoCl.sub.2.6H.sub.2 O, 
0.3 g/L Na.sub.2 MoO.sub.4.2H.sub.2 O, 2.73 g/L MnCl.sub.2, 6.6 g/L 
CaCl.sub.2.2H.sub.2 O, 1.5 g/L Cu(II)SO.sub.4.5H.sub.2 O, and 15 ml/L 85% 
H.sub.3 PO.sub.4. In addition, the following components were added to the 
media after sterilization to achieve the final concentrations indicated: 
10 mg/L tetracycline and 320 mg/L thiamine. Polypropylene glycol 2000 was 
added if a foaming problem was observed. 
Fermentor (100 L volume) 
2000 mL of the Fermentor Inoculum was then aseptically transferred to a 
100-liter BioLafitte fermentor containing 54 L of DM59 medium described 
above. 
The fermentor was run at 30.+-.1.degree. C., controlling dissolved oxygen 
at 20% and glucose between 0-6 g/L. At OD 30.+-.2, induction occurred by 
lowering the temperature of the fermentor to 26.degree. C., adding 43.5 mL 
of 100 mM IPTG and 73 mL of 50 mg/mL hemin. At 3 hours post induction, 96 
mL of 50 mg/mL hemin was added, at 6 hours post induction, 125 mL of 50 
mg/mL hemin was added, at 9 hours post induction, 125 mL of 50 mg/mL hemin 
was added and at 12 hours post induction, 125 mL of 50 mg/mL hemin was 
added. Harvest and further purification occurred at 16 hours post 
induction. Cells were either immediately purified or frozen for later 
purification. 
Purification 
If required, frozen cells were partially thawed in warm water for 
approximately 20-30 minutes. Cells were chopped into small bits in a steel 
beaker using break buffer (40 mM Tris base, 1 mM benzamidine) as needed. 
The chopped cells and break buffer at a ratio of 2 mL break buffer per 1 
gram of frozen cells were placed in a Waring Industrial Blender and 
homogenized for 1-5 minutes on the low setting. The solution was allowed 
to settle for 5 minutes after homogenization and any foamed material was 
removed. 
A Niro Panda.TM. cell disruption device (Niro Hudson, Inc. Hudson, Wis.) 
was prepared for homogenization by passing 200-300 mL of break buffer 
through the system. Cells were lysed by one or two passages of the 
homogenized cell solution through the Niro set at 850 bar. The pH of the 
lysate was adjusted to approximately 8 with sodium hydroxide, and 
sufficient Zn(OAc).sub.2 was added to make the solution 2-4 mM in 
Zn(OAc).sub.2. The solution was then spun at 10,000 rpm in a JA-10 rotor 
at 4.degree. C. for 60 minutes in a Beckman centrifuge. The supernatant 
was collected and was optionally diluted 1:1 with distilled water. When 
using this protocol to purify K158, care should be taken to keep levels of 
oxygen as low as possible. 
Chromatography 
All solutions were 4.degree. C. and were adjusted to the correct pH at 
4.degree. C. 500 mL of Chelating SEPHAROSE fast flow resin (Pharmacia, 
Piscataway, N.J.) was prepared by washing with 4 column volumes of 
distilled water. Flow through the column for all steps was 200 mL/min. The 
resin was charged with 2 to 3 column volumes of 2 mM Zn(OAc).sub.2 
followed by 2-3 column volumes of 200 mM NaCI. The lysate was loaded onto 
the column and washed with 4 to 6 column volumes of 20 mM Tris, 500 mM 
NaCl, pH 8.5, 7-8 column volumes of 240 mM Tris, pH 8.5, and 7-8 column 
volumes of 20 mM Tris, pH 8.5. Hemoglobin was eluted with 15 mM EDTA, 20 
mM Tris, pH 8.5 and collected into 200 mL of well oxygenated 20 mM Tris, 
pH 8.5. The column was then rinsed with an additional 3-4 column volumes 
of 15 mM EDTA, 20 mM Tris, pH 8.5, regenerated with 4 column volumes of 
200 mM NaCl and stored in 0.2 N NaOH. 
The solution was then buffer exchanged 5 times into 20 mM Tris, pH 8.5 
prior to loading onto 200 mL of a SEPHAROSE Q column. The column had been 
prepared by rinsing with 4 column volumes of distilled water, 4 column 
volumes of 1 M NaCl, 4 additional column volumes of distilled water and 
equilibrating with 3 to 4 column volumes of 20 mM Tris, pH 8.5. After 
loading the sample, the column was washed with 2 to 3 column volumes of 20 
mM Tris, pH 8.5 and eluted with 20 mM Tris, pH 7.6. Fractions were 
collected and pooled if the A.sub.575 /A.sub.540 ratio was greater than or 
equal to 1.03. The column was then cleaned with 3-4 column volumes of 1 M 
NaCl, 4 column volumes of distilled water, 2-3 column volumes of 50% 
acetic add, 4 column volumes of distilled water and finally 2-3 column 
volumes of 0.2 N NaOH for storage. The column was run at 30 mL/min flow 
rate. The resultant hemoglobin was stored at -80.degree. C. or in liquid 
nitrogen. 
EXAMPLE 2 
Construction of Di-dialpha Gene Construct Linked by a 7 Amino Acid Linker 
(SGE 939) 
A. Construction of pTZ19U/705 Mutants 
rHb1.1 genes were cloned as a BamHI/HindIII DNA fragment into pTZ19U 
(BioRad, Hercules, Calif.). This construct was then transformed using a 
modified process of the Hanahan protocol (Hanahan, J. Mol. Biol., 166:557 
(1983)) into CJ236 E. coli strain (BioRad). The Hanahan transformation 
buffer contained 45 mM MnCl.sub.2, 60 mM CaCl.sub.2, 40 mM KOAc, 620 mM 
sucrose, 15% glycerol and 100 mM rubidium chloride. A 5 ml culture of an 
E. coli strain was started in 2.times. TY broth from an isolated colony 
and cultured overnight. Then, 200 ml of 2.times. TY broth was inoculated 
with 2 ml of the overnight culture and incubated at 37.degree. C. with 
vigorous shaking for 2.5 hours. The culture was then transferred to two 
300 ml centrifuge tubes and placed on ice for 15 minutes. Cells were 
pelleted in a centrifuge at 8000 rpm, 4.degree. C., for 10 minutes and the 
supernatant was poured off. The cells were gently but thoroughly 
resuspended in 80 ml transformation buffer. The cells were again pelleted 
at 8000 rpm, 10 minutes at 4.degree. C. The cells were gently resuspended 
in 20 ml of ice-cold transformation buffer and left on ice for 30-60 
minutes. Cells were aliquoted in buffer into twenty 1 ml tubes. The cells 
were quickly frozen on dry ice and stored at -80.degree. C. 
Single-stranded DNA containing uracil substitutions was isolated and 
oligonucleotide-directed mutagenesis was performed using the Muta-gene Kit 
(BioRad, Hercules, Calif.) and standard protocols according to the 
manufacturer's instructions. Two pTZ19U/705 clones were prepared as 
follows. 
The first pTZ19U/705 clone was prepared using oligonucleotide JD29 (ACC GTT 
CTG ACT AGT AAA TAC CGT TAA TGA [SEQ. ID. NO. 18]). This oligonucleotide 
created a unique SpeI site in the end of the dialpha domains. A second 
pTZ19U/705 done was prepared using oligonudeotides JD28 (5'-GGA GGT TAA 
TTA ATG CTG TCT CCT GCA GAT-3' [SEQ. ID. NO. 19]) and JD30 (5'-CTG GTG GGT 
AAA GTT CTG GTT TGC GTT CTG-3' [SEQ. ID. NO. 20]). The resulting clone 
incorporated a unique PstI site in the dialpha genes and removed an SpeI 
site in the beta domain. 
B. Assembly of the Di-dialpha Gene Construct 
The assembly of di-dialpha gene construct was accomplished by removal of a 
dialpha gene cassette from the first pTZ19U/705 clone using BamHI/SpeI 
enzymes and gel purification of the DNA fragment. A second pTZ19U/705 
clone was cut with PstI/BglII enzymes to give a second dialpha gene 
cassette with the 5' end of the beta gene, which was also purified. These 
were then further ligated together with annealed oligonudeotides JA113 and 
JA114 to create a di-dialpha cassette with a 7 amino acid fusion peptide 
linker linking the two dialpha globins. 
JA113: 5'-CT AGT AAA TAC CGA TCG GGT GGC TCT GGC GGT TCT GTT CTG TCT CCT 
GCA-3' (SEQ.ID.NO.21). 
- JA114: 
5'-GG AGA 
CAG AAC 
AGA ACC 
GCC AGA 
GCC ACC 
CGA TCG 
GTA TTT 
A-3' 
(SEQ.ID.NO. 
22). 
This di-dialpha cassette was then ligated as a BamHI/BglII fragment into 
pSGE705 (described in PCT publication number WO 95/14038, herein 
incorporated by reference) that had the rHb1.1 genes removed as a 
BamHI/BglII fragment. The resulting di-dialpha plasmid (pSGE1000) was 
transformed into SGE1661 (also described in PCT publication number WO 
95/14038) using the modified Hanahan's protocol described above to create 
SGE939. Two other plasmids were also constructed using the same methods 
described above, pSGE1006 and pSGE1008. pSGE1006 corresponds to pSGE1000, 
except that the linker linking the two dialpha regions was excised as an 
SpeI/PstI fragment and replaced with a synthesized region encoding a 14 
amino add linker of the following sequence: 
(SEQ.ID.NO.2) 
GlyGlySerGlyGlySerGlyGlySerGlyGlySerGlyGly 
pSGE1008 was created in the same fashion as pSGE1006, except that the 
replacement linker was a 16 amino add linker of the following sequence: 
(SEQ.ID.NO.3) 
SerGlyGlySerGlyGlySerGlyGlySerGlyGlySerGlyGlySer 
EXAMPLE 3 
Construction of a High Copy Plasmid 
The construction of pSGE720 was performed in two stages. First, the pUC 
origin of replication was introduced to create plasmid pSGE715, which is 
similar to pSGE705 in that it includes the lacI gene. Then, the lacI gene 
was deleted from the plasmid and replaced with a short oligonucleotide 
containing several convenient restriction sites to create plasmid pSGE720. 
A. Construction of pSGE715 
The pUC origin of replication was introduced to create plasmid pSGE715 
through pSGE508, which is identical to pSGE509 with the exception of a 
single basepair substitution at base 602 (G.fwdarw.A). The substitution 
changes the pBR322 origin of replication to a pUC19 origin of replication. 
Plasmids pSGE508 and pSGE705 were digested to completion with restriction 
enzymes BamHI and HindIII, according to the manufacturer's instructions 
(New England Biolabs.). The plasmid, pSGE508, was digested first with 
BamHI to completion, then HindIII was added, and the digestion continued. 
The pSGE705 digest was purified with Promega Magic DNA Clean-up protocols 
and reagents (Promega, Madison, Wis.) and further digested to completion 
with BglI, according to the manufacturer's instructions (New England 
Biolabs). The enzymes in both pSGE508 and pSGE705 digests were inactivated 
by heating at 67.degree. C. for 10 minutes, then the DNA was pooled and 
purified together using Promega Magic DNA Clean-up protocols and reagents. 
The DNA was suspended in ligation buffer, T4 DNA ligase was added to one 
aliquot, and the DNA was incubated overnight at 16.degree. C. SGE1661 
cells were made competent by the method of Hanahan, using rubidium 
chloride (Hanahan, D., In DNA Cloning; A Practical Approach (Glover, D. 
M., ed.) vol. 1, pp.109-135, IRL Press, Oxford, 1985), and transformed 
with the ligation mix according to the Hanahan protocol. Transformants 
were selected by plating the cells on LB plates containing 15 .mu.g/ml 
tetracycline. Candidates were screened by restriction digestion to 
determine the presence of the rHb1.1 genes, and sequencing to detect the 
pUC origin of replication. Several candidates were identified, and the 
resulting plasmid was named pSGE715, and pSGE715 in SGE1661 was called 
SGE1453. 
The copy number of pSGE715 is about four-fold higher than pSGE705, measured 
to be about 460 plasmids per cell. As noted above, the difference between 
pSGE705 and pSGE715 is a single basepair change in the origin of 
replication region, which has been confirmed by sequencing. 
B. Construction of pSGE720 
The lacI gene was deleted from pSGE715, replacing it with a short 
oligonucleotide containing several convenient restriction sites, by the 
following steps. First, plasmid pSGE715 was digested to completion with 
restriction enzymes BamHI and NotI, according to the manufacturer's 
instructions (New England Biolabs). The pSGE715 digest was purified with 
Promega Magic DNA Clean-up protocols and reagents. The DNA was mixed with 
annealed, kinased oligonudeotides, CBG17+CBG18, and suspended in ligation 
buffer. 
CBG17 = 5'-GGCCGCCTTAAGTACCCGGGTTTCTGCAGAAAGCCCGCCTA 
(SEQ.ID.NO.: 23) 
ATGAGCGGGCTTTTTTTTCCTTAGGG-3' 
- CBG18 = 5'-GATCCCCTAAGGAAAAAAAAGCCCGCTCATTAGGCGGGCTTT (SEQ.ID.NO.: 
24) 
CTGCAGAAACCCGGGTACTTAAGGC-3' 
T4 DNA ligase was added to one aliquot, and the DNA was incubated overnight 
at 16.degree. C. SGE1821 cells were made competent by the method of 
Hanahan, using Rubidium Chloride, and transformed with the ligation mix 
according to the Hanahan protocol. SGE1821 contains pRG1 plasmids in 
addition to pSGE720. pRG1 is a low copy number plasmid containing LacIq. 
Transformants were selected by plating the cells on LB plates containing 
15 g/ml tetracycline. Candidates were screened by restriction digestion 
using PstI and Smal to detect the presence of the new linker and the 
absence of the lacI gene, and sequenced to detect the pUC origin of 
replication and the absence of the lacI gene. Several candidates were 
identified, and the resulting plasmid was named pSGE720. The plasmid, 
pSGE720 in SGE1675 was denoted SGE1464. 
EXAMPLE 4 
High Copy Di-dialpha Construct 
A second plasmid containing the di-dialpha hemoglobin genes was created 
using pSGE720 as the vector. The di-dialpha gene cassette was removed as a 
BamHI/HindIII fragment and gel purified. The vector pSGE720 was also cut 
with BamHI/HindIII and the rHb1.1 genes removed. The vector was gel 
purified. The di-dialpha cassette was ligated into the pSGE720 vector, 
resulting in a new vector pSGE1004. This new vector was then transformed 
into E. coli strain SGE1675 using the modified Hanahan method as described 
below to produce strain SGE946. 
EXAMPLE 5 
Characterization of SGE939 and SGE946 Globins 
Several 15 liter fermentations were performed on both strains SGE939 and 
SGE946 and soluble vs. insoluble western blots were performed using 
conventional methods. This data coupled with purification yields indicated 
that more soluble protein could be obtained from SGE946 (250-300 mg/L by 
the BioCAD assay (BioRad). The data obtained shows that both strains make 
di-dialpha globin and beta globin proteins, but that the SGE946 strain 
makes a larger amount of total protein and soluble protein. 
The SGE939 hemoglobin-like protein was first eluted from a Q-SEPHAROSE 
column and then from a S-SEPHAROSE column on an FPLC. Fractions were 
collected by eluting with a pH gradient. By SDS-PAGE analysis, there 
appeared to be a population of degradation products since these 
cross-reacted with anti-rHb antibodies. The cleanest fractions were pooled 
and analyzed by C4 HPLC. A chromatogram of SGE939 showed the beta globin 
eluting at 43.7 minutes as expected, and the di-dialpha peak eluting at 
61.8 minutes. Dialpha globin normally eluted at about 55 minutes under 
these conditions. There was also a peak at 56.2 minutes and a large 
shoulder on the di-dialpha peak. The peaks were collected and analyzed by 
mass spectroscopy. The beta globin peak gave the expected molecular weight 
of 15,910 daltons, while the di-dialpha peak gave a molecular weight of 
61,088 daltons. The calculated molecular weight for beta globin is 15,913, 
while the calculated molecular weight of di-dialpha globin is 61,107.8 
daltons. These results indicate that the protein expressed from SGE939 
contained the expected di-dialpha polypeptide. The protoporphyrin IX 
content was shown to be below 3%. The P.sub.50 averaged to be 24.7 and the 
n.sub.max was 1.75. 
EXAMPLE 6 
Tetra-Dialpha 
A. Construction of Di-dialpha Vector Containing the K158C Mutation 
Replacing the lysine residue at position 158 of dialpha globin allows 
chemical cross-linking of rHb1.1 molecules to form a dimeric hemoglobin 
molecule referred to as K158C. This mutation can be inserted into the 
di-dialpha expression plasmid (pSGE1000), to produce a mutant genetically 
linked di-hemoglobin that can be chemically cross-linked to form a 
tetra-hemoglobin. The modification will place the K158C mutation in the 
fourth (3'-terminal) alpha globin coding sequence of the di-dialpha 
plasmid. The K158C mutation is a 3 base change in the coding sequence, and 
can be transferred among dialpha-containing vectors on an Eag I-Bgl II 
restriction fragment. Because there are multiple Eag I sites in pSGE1000, 
an intermediate cloning step in the plasmid "pFusion II" is required. The 
cloning steps are as follows: 
1. Isolate an EagI-Bgl II fragment containing the K158C mutation from 
pSGE1.1E4 
2. Isolate large Eag I-Bgl II fragment from plasmid pFusion II, which 
removes the comparable "wild type" fragment from the second alpha gene 
3. Ligate above fragments to form the intermediate pFusion II-based vector 
containing the K158C mutation 
4. Replace the Pst I-Bgl II fragment in pSGE1000 with the Pst I-Bgl II 
fragment containing the K158C mutation. 
B. Development of a Cloning Strategy for Genetically Linked Tetra Dialpha 
Expression of a genetically linked tetra-hemoglobin molecule requires 
construction of a plasmid containing coding sequences for four dialpha 
hemoglobin genes, connected by coding sequences for peptide linkers, and 
one beta globin gene. A plasmid with these characteristics can be based on 
pSGE1000, which is currently being used to express a genetically linked 
di-hemoglobin. The following steps will be required to generate this 
plasmid: 
1. Generate a modified vector with a new restriction site at the 5' end of 
the di-dialpha coding sequence; 
2. Generate a second modified vector with a new restriction site at the 3' 
end of the di-dialpha coding sequence; 
3. Design an amino acid sequence suitable for linking the di-dialpha 
molecules in such a way that a tetra-hemoglobin can assemble and design 
the DNA sequence required to encode the peptide linker; and 
4. Assemble a new plasmid containing the two modified di-dialpha sequences, 
the linker sequence, and either a 705 or 720 plasmid background. 
Silent mutations in were identified in the di-dialpha sequence that will 
generate restriction sites unique to di-dialpha in either the 705 or 720 
(low and high-copy) plasmid backgrounds, near the 5' and 3' ends of the 
di-dialpha coding sequence. A restriction site for one of the enzymes, 
AatII, is also present in the beta globin gene; the site in the beta gene 
will be removed to facilitate cloning. A preliminary cloning strategy has 
been generated for construction of a tetra-hemoglobin expression vector as 
follows: 
1. Create an Aat II site at the 3' end of a dialpha gene in pFusion II by 
site directed mutagenesis to create a fragment denoted "A1." 
2. Subclone A1 into di-dialpha on a PstIl BglII restriction fragment to 
create "A2." 
3. Remove the AatII site from the beta globin gene in pFusion II by site 
directed mutagenesis to create "B1." 
4. Subclone "B1" into a second di-dialpha construction on a PstI/BglII 
fragment to create "B2." 
5. Create a B1pI site at the 5'end of the dialpha gene in pFusion I by site 
directed mutagenesis to create "C1." 
6. Subclone C1 on a BamHI/Spe I fragment into the modified di-dialpha 
plasmid (B2) to create "D1." 
7. Isolate the BamHI/AatII fragment from A2, and the B1pI/HindIII fragment 
from D1; ligate with a new synthetic sequence encoding a peptide linker 
containing AatII and B1PI ends, in a convenient plasmid background to form 
a tetrahemoglobin coding sequence. 
EXAMPLE 7 
General Transformation Procedure 
A modified Hanahan protocol was used to produce competent E. coli cells. 
The Hanahan Transformation buffer contains 45 mM MnCl.sub.2, 60 mM 
CaCl.sub.2, 40 mM KOAc, 620 mM sucrose, 15% glycerol and 100 mM rubidium 
chloride. A 5 ml culture of an E. coli strain was started in 2.times. TY 
broth from an isolated colony and cultured overnight Then, 200 ml of 
2.times. TY broth was inoculated with 2 ml of the overnight culture and 
incubated at 37.degree. C. with vigorous shaking for 2.5 hours. The 
culture was then transferred to two 300 ml centrifuge tubes and placed on 
ice for 15 minutes. Cells were pelleted in a centrifuge at 8000 rpm's, 
4.degree. C., for 10 minutes and the supernatant was poured off. The cells 
were resuspended gently, but thoroughly in 80 ml transformation buffer. 
The cells were again pelleted at 8000 rpm for 10 minutes at 4.degree. C. 
The cells were gently resuspended in 20 ml of ice-cold transformation 
buffer and left on ice for 30-60 minutes. Cells were aliquoted in buffer 
into twenty 1 ml tubes. The cells were quickly frozen on dry ice and 
stored at -80.degree. C. 
EXAMPLE 8 
Preparation of BMH-crosslinked di-alphaK158C (Di-hemoglobin) 
Di-hemoglobin was produced by crosslinking dialpha hemoglobin containing a 
K158C mutation in the second alpha globin domain using bismaleimidohexane 
(BMH, Pierce Chemical Co., Rockford, Ill.). BMH is a homobifunctional 
maleimide crosslinker, and its primary reactivity is towards sulfhydryl 
residues. The linkage is irreversible once formed. The alkane spacer 
between the maleimide residues is hexane (six carbons) and the molecule 
has poor solubility in buffered aqueous solutions. The nominal length of 
the crosslinker is 16.1 .ANG.. 
K158C was concentrated to 60 mg/mL in 20 mM Tris buffer pH 8, and 
deoxygenated by gas exchange with humid oxygen free nitrogen in a rotating 
glass flask (ROTOVAP RE111, Brinkmann, Inc., Cuntiague Road, Westbury, 
N.Y.). K158C was maintained in the deoxy form in order to limit the 
reaction of BMH with the intrinsic sulfhydryls of hemoglobin, especially 
residue Cys93 in the beta subunit. The reactivity of this residue with 
sulfhydryl reactive reagents is generally at least 50 fold slower in the 
deoxy form than in liganded forms of hemoglobin. The reactivity of the 
surface K158C residue is not affected significantly by the heme ligation 
state. 
A solution of BMH was prepared in pure dimethyl sulfoxide (DMSO) at 10 
mg/mL. An aliquot of this solution was added to the deoxyHb solution (0.6 
moles of BMH per mole of Hb, maintaining deoxy conditions) with swirling 
to mix, and the sample was allowed to react for 1 hour on ice. Following 
reaction, the hemoglobin solution was centrifuged or filtered (0.2 micron) 
to remove any precipitated material, diluted to 25 mg/mL and then 
chromatographed on SEPHACRYL S-200 HR (Pharmacia, Uppsala, Sweden) to 
resolve the dihemoglobin fraction from the unreacted monohemoglobin and 
the small amount of trihemoglobin formed during the reaction. Two S-200 HR 
columns (Pharmacia BPP 113, ca. 6L of resin each, 11.3 cm 
diameter.times.60 cm) were used in series to give acceptable resolution 
and volume handling capabilities. The yield of coupling was typically 60%, 
and about 50% of the starting hemoglobin was recovered following size 
exclusion chromatography. Following chromatography on Q-SEPHAROSE to 
remove endotoxin, the dihemoglobin was submitted for several routine 
analyses and the results are reported below. Methods for these analyses 
are described in PCT publication WO 95/14038. Average molecular weight was 
determined by size exclusion chromatography using a SUPEROSE 12 column 
using Bio-Rad molecular weight standards (Bio-Rad, Hercules, Calif.). 
______________________________________ 
Assay Result 
______________________________________ 
Endotoxin (LAL assay) 0.6 EU/mL 
E. coli protein below detection 
Protoporphyrin IX 1.14% 
p50, Torr at 37.degree. C. 32.7 
Nmax. 2.09 
Average molecular weight 128 kDa 
______________________________________ 
EXAMPLE 9 
LAL Assay for Endotoxin 
Fifty microliters of endotoxin standard, blank diluent, or hemoglobin 
solution (rHb1.1) was mixed with 50 ul of LAL lysate (BioWhittaker, Inc., 
Walkersville, Md.) in a well of a 96-well, pyrogen-free microtiter plate, 
according to the manufacturer's instructions. The mixture was allowed to 
incubate for 30 minutes in a 37.degree. C. water bath. One hundred 
microliters of acetate-Ile-Glu-Gly-Arg-(SEQ ID. NO.25) conjugated to 
para-nitroaniline (chromogenic substrate) was added to each well and the 
plate allowed to incubate for an additional 16 to 60 minutes at 37.degree. 
C. The reaction was stopped by the addition of 50 ul 25% glacial acetic 
acid, and the samples transferred to HPLC sample vials for analysis. 
Twenty microliters of each sample was injected onto a Vydac C4-reversed 
phase chromatography column (2 mm.times.250 mm), pre-equilibrated at 
40.degree. C., 5% Solvent B. (Solvent A is 20% acetonitrile in water with 
0.1% TFA and Solvent B is 100% acetonitrile with 0.1% TEA). The 
chromatographic system was run at a flow of 1 ml/min. Separation was 
achieved as follows: a 1 minute hold in 95% Solvent A/5% Solvent B after 
injection, a 4 minute ramp to 50% Solvent A/50% Solvent B, a 2 minute 
increase to 100% Solvent B, a 3 minute wash in 100% Solvent B, a return to 
95% Solvent A/5% Solvent B over 1 minute and an equilibration at 95% 
Solvent A/5% Solvent B for 4 minutes. The separation was monitored at 405 
nm. 
The peak areas of the standard solutions were used to construct a standard 
curve against which test samples were measured. A series of curves were 
generated from the analysis of standard solutions ranging in 
concentrations from 0.5 EU/ml to 0.0005 EU/ml. Linearity was achieved when 
the standards were analyzed in groups according to the time of incubation. 
One curve was generated from analysis of samples incubated with 
chromogenic substrate for 16 minutes, others were generated from analysis 
of samples incubated with chromogenic substrate for 30 minutes and 60 
minutes. Therefore, a standard curve for use in a particular circumstance 
depended on the sensitivity of the endotoxin measurement that was 
required. 
EXAMPLE 10 
Production of Penta-hemoglobin 
rHb1.1 containing a K158C mutation in the dialpha globin (hereinafter 
referred to as K158C) was expressed and purified as described above. 
rHb1.1 was expressed and purified as described in PCT publication number 
WO 95/14038, filed Nov. 14, 1994, entitled Purification of Hemoglobin." 
The penta-hemoglobin was then formed by reacting K158C with a core rHb1.1 
molecule (that did not contain the K158C mutation) activated as described 
below. 
The core rHb1.1 molecule was activated by reacting with sulfosuccinimidyl 
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (Pierce 
Chemicals, Rockford, Ill.). Sulfo-SMCC is a water soluble 
heterobifunctional crosslinker that reacts with both amine and sulfhydryl 
functional groups. Reaction with lysine residues on rHb1.1 leads to loss 
of the sulfosuccinimide group with the formation of a stable amide linkage 
between the protein and the succinimide moiety. The maleimide residues are 
highly reactive towards sulfhydryl groups. Therefore, following reaction 
with sulfo-SMCC, the rHb1.1 has been "activated" at multiple surface 
lysine residues towards reaction with the surface sulfhydryl residue of 
K158C. The N-(4-carboxycyclohexylmethylmaleimide) residues are 
particularly stable to hydrolysis and the "activated" rHb1.1 can thus be 
manipulated extensively prior to addition of K158C. 
The desired extent of modification of rHb1.1 was determined empirically by 
reaction with K158C following activation. The initial reaction with 
sulfo-SMCC was modulated by altering the concentration of sulfo-SMCC and 
reaction time, until a covalent Hb polymer of the desired size range was 
achieved upon subsequent reaction with K158C. Once determined, these 
conditions were used throughout. In determining these conditions, the 
stability of the polymer was monitored. 
To activate the rHb1.1 that formed the core of the penta-hemoglobin 
molecule, a solution of sulfo-SMCC, 10 mg/mL in 100 mM sodium borate 
buffer pH 8.5, was added to a solution of rHb1.1 (30 mg/mL) at a molar 
ratio of 35:1 under oxy conditions at 22.degree. C. This was allowed to 
react for 35 minutes with gentle mixing. The succinimide reactive portion 
of the crosslinker was then quenched by addition of glycine at a molar 
ratio of 25:1 (25 M glycine to 1 M crosslnker). 
The reaction mixture was then chromatographed on Sephadex G25 equilibrated 
in 50 mM Tris-HCl buffer, pH 8.0 to remove quenched crosslinker and borate 
ions and to buffer exchange the activated rHb1.1. Following buffer 
exchange, the activated rHb1.1 was concentrated to 15 mg/mL and converted 
into the deoxy form by deoxygenating on a rotary evaporator (ROTOVAP 
RE111, Brinkmann, Inc., Cuntiague Road, Westbury, N.Y.) flushed with 
humidified nitrogen. 
Following activation of the rHb1.1 molecule, penta-hemoglobin was 
synthesized as follows. Activated hemoglobin was concentrated to 15 mg/mL 
and deoxygenated as above. Deoxy activated rHb1.1 was added to previously 
deoxygenated K158C (60 mg/ml) at a molar ratio of 1 to 5.5 in drop wise 
fashion. Crosslinking was allowed to proceed spontaneously at room 
temperature (22.degree. C.) for 3 hours with gentle mixing. The mixture 
was then cooled to 4.degree. C. and cysteine was added to a final 
concentration of 8.5 mM and allowed to react for 15 minutes to quench the 
maleimide portion of the crosslinker. The resultant product contained a 
mixture of monohemoglobin, dihemoglobin, trihemoglobin, tetrahemoglobin, 
pentahemoglobin, hexahemoglobin and higher order multimers. Approximately 
20% of the mixture was mono- and di-hemoglobin. Approximately 65% of the 
mixture was most likely tetra-, penta- and hexa-hemoglobin. 
The desired molecular weight fraction was resolved from the mixture by size 
exclusion chromatography on Sephacryl S-200 HR and S-300 HR. Two columns 
(Pharmacia BPP113, 60 cm length, one of each resin type) were used in 
series to achieve the desired fractionation. Both columns were 
equilibrated with phosphate buffered saline, pH 7.5. Alternatively, the 
molecular weight fractions were separated using ion exchange 
chromatography as described below. 
The pentaHb fraction exhibited the following equilibrium oxygen binding 
properties: P.sub.50 =32 Torr and N.sub.max =2.1 on average for multiple 
determinations. 
EXAMPLE 11 
Formation of Penta-hemoglobin Using Sulfo-GMBS Crosslinker 
Sulfo-GMBS (N-.gamma.-maleimidobutyrloxy)succinimide ester) was dissolved 
at 10 mg/mi in 100 mM sodium borate, pH 8.5. All other steps were 
performed identically to the steps disclosed in example 10. The final 
penta hemoglobin with GMBS crosslinking was produced in approximately the 
same yield as in example 10. 
The pentaHb fraction produced using the GMBS linker exhibited the following 
equilibrium oxygen binding properties: P.sub.50 =30 Torr and N.sub.max 
=2.1 for multiple determinations. 
EXAMPLE 12 
Formation of Penta-hemoglobin-K158C Core 
An entire multimeric Hb-like protein can be prepared using only K158C 
tetramers. The procedure described in Example 10 can be followed 
identically. Excess crosslinker is removed by, for example, gel filtration 
or tangential flow ultrafiltration in the continued presence of borate 
buffer. Borate buffer should be maintained while sulfhydryl reactive 
crosslinker is being removed. Following adequate removal of the (amine) 
quenched crosslinker, the borate buffer is exchanged completely for 
another suitable buffer, such as Tris-HCl buffer (using, for example, gel 
filtration or tangential flow ultrafiltration). This readies the material 
for the final crosslinking step as described in Example 10 in which 
pentaHb is produced. 
EXAMPLE 13 
Purification of Glutaraldehyde Crosslinked Hemoglobin by Anion Exchange 
Chromatography 
Recombinant hemoglobin (rHb1.1) was expressed, prepared and purified as 
described in PCT publication number WO 95/14038, filed Nov. 14, 1994, 
entitled "Purification of Hemoglobin" herein incorporated by reference in 
its entirety. This hemoglobin (24 g) was concentrated to .about.150 mg/ml 
and deoxygenated in a 1L round bottom flask by purging for 5 hours with 
humidified nitrogen on a Brinkmann ROTOVAP RE111 (Brinkmann, Inc., 
Cuntiague Road, Westbury, N.Y.). and crosslinked at 25.degree. C. with a 
6:1 molar ratio of glutaraldehyde:rHb1.1 (glutaraldehyde was a 10% aqueous 
solution, diluted from 25% aqueous solution, Sigma Chemical Company, St. 
Louis, Mo.). The reaction was terminated after 4 minutes with a 3:1 molar 
ratio of sodium borohydride: glutaraldehyde then buffer exchanged with 
ultrafiltration into a 20 mM Tris, pH 8.9 (8.degree. C.) buffer. The 
crosslinked hemoglobin (21 g) was then loaded onto a 450 ml SEPHAROSE-Q 
ion-exchange resin. After the column was loaded it was washed 8 CV's of 20 
mM Tris, pH 7.6 (8.degree. C.) followed by elution with 20 mM Tris, pH 7.4 
(8.degree. C.). 
TABLE 3 
______________________________________ 
Protein distribution displayed as % of total in eluted fraction 
65 kDa 128 kDa 190 kDa 
&gt;230 kDa 
______________________________________ 
Load 27.7 17.7 13.3 40.5 
(230-5000 kDa) 
Breakthrough 99.3 
(200-5000 kDa) 
pH 7.6 Wash 60.9 25.6 7.1 2.5 
(250-535 kDa) 
pH 7.4 wash 1.0 6.6 13.1 79.2 
(250-2000 kDa) 
peak = 410 kDa 
______________________________________ 
EXAMPLE 14 
Selective Purification of Glutaraldehyde Crosslinked Hemoglobin Molecular 
Weight Fractions Using pH Elution 
Glutaraldehyde crosslinked hemoglobin (.about.1 g) prepared as described in 
Example 13 was loaded onto a 50 ml bed volume SEPHAROSE-Q ion-exchange 
resin. The column was washed with loading buffer as described in the 
previous example followed by elution of the bound protein with a stepwise 
pH gradient beginning with a 20 mM Tris buffer, pH 7.8 (8.degree. C.). The 
pH steps were decreased in 0.1 pH unit increments with only selected 
fractions shown here for illustration. The use of very small pH increments 
improved resolution of the different molecular weight fractions. 
TABLE 4 
______________________________________ 
Protein distribution displayed as % of total in eluted fraction 
65 kDa 128 kDa 190 kDa 
&gt;230 kDa 
______________________________________ 
Load 30.7 19.0 13.5 36.7 
(260-4200 kDa) 
Load Break 98.0 
(pH 8.9) (260-4200 kDa) 
peak = 1400 kDa 
pH 7.8 wash 92.6 5.7 
pH 7.7 wash 38.8 54.6 5.1 
pH 7.5 wash 7.4 20.3 29.4 41.7 
(250-2000 kDa) 
peak = 285 kDa 
pH 7.3 wash 3.5 8.2 11.2 76.6 
(250-4200 kDa) 
peak = 453 kDa 
______________________________________ 
EXAMPLE 15 
Effect of Protein Concentration on Separation Efficiency 
Hemoglobin was crosslinked as described in Example 13 and loaded onto a 50 
ml bed volume SEPHAROSE-Q ion-exchange resin. The column loads were 
sequentially increased (Table Five). Loading procedures and elution of the 
protein was the same as in Example 13. As noted in Table Five below, 
increasing the hemoglobin load improved the efficiency of separation, 
particularly in the region of 230-800 kDa molecular weights. 
TABLE 5 
______________________________________ 
Protein distribution displayed as % of total protein in eluted fraction 
230-800 
65 kDa 128 kDa 190 kDa kDa &gt;800 
kDa 
______________________________________ 
Load 31.6 19.0 13.8 24.8 10.7 
10 g/L resin 2.7 6.6 9.3 50.1 31.2 
25 g/L resin 2.6 7.3 9.9 54.2 25.9 
50 g/L resin 1.9 7.8 11.8 57.7 20.6 
80 g/L resin 1.2 6.1 11.4 57.2 23.9 
______________________________________ 
EXAMPLE 16 
Effect of Column Size on Separation 
Hemoglobin was crosslinked as described in Example 13 and loaded onto 
either a 50 ml or 2100 ml bed volume SEPHAROSE-Q ion-exchange resin. The 
column loads were 30 g/L resin and 14.7 g/L resin for the 50 ml and 2100 
ml columns respectively. Protein distributions in each column load were 
similar to those described previously. Loading and elution of the protein 
was the same as in Example 14. As noted in Table Six below, there was only 
a minimal effect of column size on efficiency of separation. Therefore, 
this methodology can be applicable to any scale of separation. 
TABLE 6 
______________________________________ 
Protein distribution displayed as % of total protein in eluted fraction 
65 kDa 128 kDa 190 kDa 
&gt;230 kDa 
______________________________________ 
50 ml 2.6 9.7 15.1 72.3 
Q-SEPHAROSE (250-4200 kDa) 
column peak = 328 kDa 
2.1 L 3.9 9.1 13.0 73.6 
Q-SEPHAROSE (250-4200 kDa) 
column peak 426 kDa 
______________________________________ 
EXAMPLE 17 
Selective Purification of Pentameric Hemoglobin Molecular Weight Fractions 
By Ion Exchange 
Super Q 650 C (TosoHaas) was equilibrated with 5 CV's of 20 mM Tris pH=8.9. 
The column was sized at binding of 15 grams protein per liter of resin. 
The pH and the conductivity of the protein sample were adjusted to match 
the equilibration buffer and loaded onto the column at approximately 4.5 
grams for an approximately 300 ml column. The column was then washed with 
2 column volumes of 20 mM Tris pH=8.9, followed by 7-8 CV wash 25 mM 
Bis-Tris/Tris pH=7.5, which allowed for removal of monomeric hemoglobin. 
The column was eluted using 25 mM Bis-Tris/Tris, 100 mM NaCl pH=7.5. After 
this purification, only approximately 3% monomeric hemoglobin remained in 
the purified pentahemoglobin solution, indicating a 5-6 fold purification 
across the anion exchange step. 
The foregoing description of the invention is exemplary for purposes of 
illustration and explanation. It will be apparent to those skilled in the 
art that changes and modifications are possible without departing from the 
spirit and scope of the invention. It is intended that the following 
claims be interpreted to embrace all such changes and modifications. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 25 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 7 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence :Peptide 
linker to couple dialpha domains 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence :Peptide 
linker to couple dialpha domains 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Peptide 
linker to couple dialpha domains 
- - &lt;400&gt; SEQUENCE: 1 
- - Ser Gly Gly Ser Gly Gly Ser 
1 5 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 14 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence :Peptide 
linker to couple dialpha domains 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Peptide 
linker to couple dialpha domains 
- - &lt;400&gt; SEQUENCE: 2 
- - Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gl - #y Ser Gly Gly 
1 5 - # 10 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 16 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence :Peptide 
linker to couple dialpha domains 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Peptide 
linker to couple dialpha domains 
- - &lt;400&gt; SEQUENCE: 3 
- - Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gl - #y Gly Ser Gly Gly Ser 
1 5 - # 10 - # 15 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 45 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 3' primer for - #generation of a 
alpha gene with Xba I site 
- - &lt;400&gt; SEQUENCE: 4 
- - cgggaatacg gtctagatca ttaacggtat ttcgaagtca gaacg - # 
- #45 
- - - - &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 95 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for genera - #tion of tac 
promotor with Bam HI-EagI site 
- - &lt;400&gt; SEQUENCE: 5 
- - gatccgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tg - 
#acggataa 60 
- - caatttcaca caggaaatta attaatgctg tctcc - # 
- # 95 
- - - - &lt;210&gt; SEQ ID NO 6 
&lt;211&gt; LENGTH: 96 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for genera - #tion of tac 
promotor with Bam HI-EagI site 
- - &lt;400&gt; SEQUENCE: 6 
- - ggccggagac agcattaatt aatttcctgt gtgaaattgt tatccgctca ca - 
#attccaca 60 
- - cattatacga gccgatgatt aattgtcaac agctcg - # 
- # 96 
- - - - &lt;210&gt; SEQ ID NO 7 
&lt;211&gt; LENGTH: 64 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 5' primer for - #generation of an 
alpha gene with EcoRI, BamHI and - #EagI sites 
- - &lt;400&gt; SEQUENCE: 7 
- - tcggattcga attccaagct gttggatcct tagattgaac tgtctccggc cg - 
#ataaaacc 60 
- - accg - # - # - # 
64 
- - - - &lt;210&gt; SEQ ID NO 8 
&lt;211&gt; LENGTH: 55 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 5' primer for - #generation of a 
beta gene with Xba site 
- - &lt;400&gt; SEQUENCE: 8 
- - cggaagccca atctagagga aataatatat gcacctgact ccggaagaaa aa - #tcc 
55 
- - - - &lt;210&gt; SEQ ID NO 9 
&lt;211&gt; LENGTH: 44 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 3' primer for - #generation of a 
beta gene with Hind III site 
- - &lt;400&gt; SEQUENCE: 9 
- - cccgaaacca agcttcatta gtgagctagc gcgttagcaa cacc - # 
- # 44 
- - - - &lt;210&gt; SEQ ID NO 10 
&lt;211&gt; LENGTH: 37 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or reverse primer fo - #r mutagenesis 
in a beta gene 
- - &lt;400&gt; SEQUENCE: 10 
- - tttaagctta attagtggta tttgtgagct agcgcgt - # 
- # 37 
- - - - &lt;210&gt; SEQ ID NO 11 
&lt;211&gt; LENGTH: 37 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or reverse primer fo - #r mutagenesis 
in a alpha gene 
- - &lt;400&gt; SEQUENCE: 11 
- - cagcattaat taacctcctt agtgaaattg ttatccg - # 
- # 37 
- - - - &lt;210&gt; SEQ ID NO 12 
&lt;211&gt; LENGTH: 45 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or reverse primer fo - #r mutagenesis 
in a beta gene 
- - &lt;400&gt; SEQUENCE: 12 
- - ggtgcatata tttacctcct tatctagatc attaacggta tttcg - # 
- #45 
- - - - &lt;210&gt; SEQ ID NO 13 
&lt;211&gt; LENGTH: 10 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for genera - #tion of a Pme 
I linker in pSelect-1 
- - &lt;400&gt; SEQUENCE: 13 
- - ggtttaaacc - # - # 
- # 10 
- - - - &lt;210&gt; SEQ ID NO 14 
&lt;211&gt; LENGTH: 58 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 5' primer for - #production of a 
lacI gene with Hind III and No - #t I sites 
- - &lt;400&gt; SEQUENCE: 14 
- - ggcgaataaa agcttgcggc cgcgttgaca ccatcgaatg gcgcaaaacc tt - #tcgcgg 
58 
- - - - &lt;210&gt; SEQ ID NO 15 
&lt;211&gt; LENGTH: 69 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 3' primer for - #production of a 
IacI gene with bam HI site 
- - &lt;400&gt; SEQUENCE: 15 
- - gggcaaatag gatccaaaaa aaagcccgct cattaggcgg gctttatcac tg - 
#cccgcttt 60 
- - ccagtcggg - # - # 
- # 69 
- - - - &lt;210&gt; SEQ ID NO 16 
&lt;211&gt; LENGTH: 54 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 5' primer for - #production of a 
pBR332 ori with Bam HI and Not - # I sites 
- - &lt;400&gt; SEQUENCE: 16 
- - ccccgaaaag gatccaagta gccggcggcc gcgttccact gagcgtcaga cc - #cc 
54 
- - - - &lt;210&gt; SEQ ID NO 17 
&lt;211&gt; LENGTH: 42 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or 3' primer for - #generation of 
pBR332 ori with PmeI site 
- - &lt;400&gt; SEQUENCE: 17 
- - ggcggtcctg tttaaacgct gcgctcggtc gttcggctgc gg - # 
- # 42 
- - - - &lt;210&gt; SEQ ID NO 18 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for introd - #uction of SpeI 
site in the end of dialpha dom - #ains 
- - &lt;400&gt; SEQUENCE: 18 
- - accgttctga ctagtaaata ccgttaatga - # - # 
30 
- - - - &lt;210&gt; SEQ ID NO 19 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for introd - #uction of Pst 
I site in dialpha genes and fo - #r removal of SpeI 
site in a beta domain 
- - &lt;400&gt; SEQUENCE: 19 
- - ggaggttaat taatgctgtc tcctgcagat - # - # 
30 
- - - - &lt;210&gt; SEQ ID NO 20 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for introd - #uction of Pst 
I site in dialpha genes and fo - #r removal of SpeI 
site in a beta domain 
- - &lt;400&gt; SEQUENCE: 20 
- - ctggtgggta aagttctggt ttgcgttctg - # - # 
30 
- - - - &lt;210&gt; SEQ ID NO 21 
&lt;211&gt; LENGTH: 50 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for introd - #uction of 7 
amino acid fusion peptide linker - #connecting two 
dialpha globins 
- - &lt;400&gt; SEQUENCE: 21 
- - ctagtaaata ccgatcgggt ggctctggcg gttctgttct gtctcctgca - # 
50 
- - - - &lt;210&gt; SEQ ID NO 22 
&lt;211&gt; LENGTH: 42 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide or primer for introd - #uction of 7 
amino acid fusion peptide linker - #connecting two 
dialpha globins 
- - &lt;400&gt; SEQUENCE: 22 
- - ggagacagaa cagaaccgcc agagccaccc gatcggtatt ta - # 
- # 42 
- - - - &lt;210&gt; SEQ ID NO 23 
&lt;211&gt; LENGTH: 67 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide for introduction of - #convenient 
restriction sites in place of a - #deleted lacI gene 
- - &lt;400&gt; SEQUENCE: 23 
- - ggccgcctta agtacccggg tttctgcaga aagcccgcct aatgagcggg ct - 
#tttttttc 60 
- - cttaggg - # - # 
- # 67 
- - - - &lt;210&gt; SEQ ID NO 24 
&lt;211&gt; LENGTH: 67 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: 
Oligonucleotide for introduction of - #convenient 
restriction sites in place of a - #deleted lacI gene 
- - &lt;400&gt; SEQUENCE: 24 
- - gatcccctaa ggaaaaaaaa gcccgctcat taggcgggct ttctgcagaa ac - 
#ccgggtac 60 
- - ttaaggc - # - # 
- # 67 
- - - - &lt;210&gt; SEQ ID NO 25 
&lt;211&gt; LENGTH: 4 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Ile is acetate - Ile 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: peptide 
reagent 
- - &lt;400&gt; SEQUENCE: 25 
- - Ile Glu Gly Arg 
__________________________________________________________________________