U.S. patent application number 16/777932 was filed with the patent office on 2020-08-06 for recombinant hemoglobins and methods of preparation and use thereof.
The applicant listed for this patent is Cheer Global Limited. Invention is credited to Sui Yi KWOK, Norman Fung Man WAI, Terence Shau Yin WAI, Shan YU.
Application Number | 20200247877 16/777932 |
Document ID | / |
Family ID | 1000004627378 |
Filed Date | 2020-08-06 |
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United States Patent
Application |
20200247877 |
Kind Code |
A1 |
KWOK; Sui Yi ; et
al. |
August 6, 2020 |
Recombinant Hemoglobins and Methods of Preparation and Use
Thereof
Abstract
The present disclosure relates to recombinant hemoglobins with
improved oxygen carrying capacity and methods of preparation and
use thereof. The present disclosure also relates to a
pharmaceutical composition including the recombinant hemoglobin
useful for treating oxygen deficiency related diseases or
conditions, such as stroke, hemorrhagic shock, peripheral arterial
disease (PAD), acute mountain sickness (AMS), cancer, and
Parkinson's disease (PD).
Inventors: |
KWOK; Sui Yi; (Hong Kong,
CN) ; WAI; Norman Fung Man; (Vancouver, CA) ;
YU; Shan; (Hong Kong, CN) ; WAI; Terence Shau
Yin; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cheer Global Limited |
Hong Kong |
|
CN |
|
|
Family ID: |
1000004627378 |
Appl. No.: |
16/777932 |
Filed: |
January 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62799829 |
Feb 1, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/805 20130101;
C12N 1/20 20130101; C12P 21/00 20130101 |
International
Class: |
C07K 14/805 20060101
C07K014/805; C12P 21/00 20060101 C12P021/00; C12N 1/20 20060101
C12N001/20 |
Claims
1. A recombinant hemoglobin comprising a di-alpha chain and two
beta chains, wherein the di-alpha chain comprises a polypeptide
sequence having at least 98.93% sequence homology with SEQ ID NO:
1, wherein the amino acids at position 1 and position 143 of SEQ ID
NO: 1 must be methionine, the amino acids at position 29 and
position 171 of SEQ ID NO: 1 must be phenylalanine, and the amino
acids at position 58 and position 200 of SEQ ID NO: 1 must be
glutamine.
2. The recombinant hemoglobin of claim 1, wherein the di-alpha
chain comprises a polypeptide sequence having at least 99.29%
sequence homology with SEQ ID NO: 1.
3. The recombinant hemoglobin of claim 2, wherein the di-alpha
chain comprises a polypeptide having at least 99.64% sequence
homology with SEQ ID NO: 1.
4. The recombinant hemoglobin of claim 3, wherein the di-alpha
chain is a polypeptide sequence having SEQ ID NO: 1.
5. The recombinant hemoglobin of claim 1, wherein each of the two
beta chains comprises a polypeptide sequence having at least 97.94%
sequence homology with SEQ ID NO: 2, wherein the amino acid at
position 1 must be methionine.
6. The recombinant hemoglobin of claim 5, wherein each of the two
beta chains is a polypeptide sequence having SEQ ID NO: 2.
7. The recombinant hemoglobin of claim 1, wherein each of the two
beta chains comprises a polypeptide sequence having at least 97.94%
sequence homology with SEQ ID NO: 3, wherein the amino acid at
position 1 must be methionine, the amino acid at position 82 must
be aspartic acid, and the amino acid at position 108 must be
lysine.
8. The recombinant hemoglobin of claim 7, wherein each of the two
beta chains comprises a polypeptide sequence having at least 98.63%
sequence homology with SEQ ID NO: 3.
9. The recombinant hemoglobin of claim 8, wherein each of the two
beta chains comprises a polypeptide sequence having at least 99.31%
sequence homology with SEQ ID NO: 3.
10. The recombinant hemoglobin of claim 9, wherein each of the two
beta chains comprises a polypeptide sequence of SEQ ID NO: 3.
11. A pharmaceutical composition comprising the recombinant
hemoglobin of claim 1 and at least one pharmaceutically acceptable
carrier.
12. A method of treating an oxygen deficiency related disease in a
subject in need thereof comprising the step of administering a
therapeutically effective amount of the pharmaceutical composition
of claim 11 to the subject.
13. The method of claim 12, wherein the oxygen deficiency related
disease comprises cancer, stroke, hemorrhagic shock, acute mountain
sickness (AMS), peripheral arterial disease (PAD), or Parkinson's
disease (PD).
14. A method of producing the recombinant hemoglobin of claim 1,
comprising the steps of: (a) providing a host cell comprising a
polynucleotide encoding the di-alpha chain and a polynucleotide
encoding a beta chain; and (b) inducing the host cell containing
the polynucleotide encoding the di-alpha chain and the
polynucleotide encoding the beta chain to express recombinant
hemoglobin thereby producing the recombinant hemoglobin of claim
1.
15. The method of claim 14, wherein the polynucleotide encoding the
di-alpha chain and the polynucleotide encoding the beta chain
comprise a polynucleotide sequence of SEQ ID NO: 4 and a
polynucleotide sequence of SEQ ID NO: 5, respectively.
16. The method of claim 14, wherein the polynucleotide encoding the
di-alpha chain and the polynucleotide encoding the beta chain
comprises a polynucleotide sequence of SEQ ID NO: 4 and a
polynucleotide sequence of SEQ ID NO: 6, respectively.
17. The method of claim 14, wherein the host cell is selected from
the group consisting of JM109 E. coli bacterial strain with lambda
DE3, BL21-AI E. coli bacterial strain without lambda DE3, and
SHuffle E. coli bacterial strain without lambda DE3.
18. The method of claim 14, wherein the host cell further comprises
a polynucleotide encoding HemH.
19. The method of claim 14, wherein the host cell further comprises
a polynucleotide encoding a Heme-transporter.
20. The method of claim 14 further comprising the steps of: (a)
rupturing the host cell after the step of inducing the host cell to
express recombinant hemoglobin to obtain a solution comprising the
recombinant hemoglobin; and (b) purifying the recombinant
hemoglobin to obtain a purified recombinant hemoglobin.
21. A system for producing the recombinant hemoglobin of claim 1,
the system comprising: an Escherichia coli or non-Escherichia coli
host cell comprising: a polynucleotide encoding HemH; a
polynucleotide encoding a Heme-transporter; a polynucleotide
encoding the di-alpha chain, and a polynucleotide encoding the beta
chain, wherein the di-alpha chain comprises a polypeptide sequence
having at least 98.93% sequence homology with SEQ ID NO: 1, wherein
the amino acids at position 1 and position 143 of SEQ ID NO: 1 must
be methionine, the amino acids at position 29 and position 171 of
SEQ ID NO: 1 must be phenylalanine, and the amino acids at position
58 and position 200 of SEQ ID NO: 1 must be glutamine; and the beta
chain comprises a polypeptide sequence having at least 99.31%
sequence homology with SEQ ID NO: 2, wherein the amino acid at
position 1 must be methionine; or a polypeptide sequence having at
least 97.94% sequence homology with SEQ ID NO: 3, wherein the amino
acid at position 1 must be methionine, the amino acid at position
82 must be aspartic acid, and the amino acid at position 108 must
be lysine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 62/799,829, filed on Feb. 1, 2019, the
contents of which are hereby incorporated by reference in their
entirety for all purposes.
TECHNICAL FIELD
[0002] The present disclosure generally relates to recombinant
hemoglobins and methods preparation, purification, and use thereof.
The present disclosure further relates to pharmaceutical
compositions comprising the recombinant hemoglobins and methods of
using the recombinant hemoglobin-based pharmaceutical composition
to treat oxygen deficiency related diseases, such stroke,
hemorrhagic shock, peripheral arterial disease (PAD) and acute
mountain sickness (AMS), and other diseases or conditions, such as
cancer and Parkinson's disease (PD).
BACKGROUND
[0003] Hemoglobin (Hb) is an oxygen-carrying protein enriched in
red blood cells (erythrocytes) that delivers oxygen to body tissues
via blood flow through the circulatory system. The oxygen-carrying
protein comprises four associated polypeptide chains (two alpha
chains and two beta chains), and bears a group known as heme whose
iron atom temporarily binds to oxygen in the lungs and releases it
throughout the body. Hemoglobin can also bind carbon monoxide (CO)
to form (carbonmonoxy)hemoglobin (HbCO), which can reduce the total
amount of Hb that is available to deliver oxygen to the body.
[0004] Hypoxia is common in cancers and can lead to ionizing
radiation and chemotherapy resistance by depriving tumor cells of
the oxygen essential for the cytotoxic activities of these agents.
Hypoxia may also reduce tumor sensitivity to radiation therapy and
chemotherapy through one or more indirect mechanisms that include
proteomic and genomic changes.
[0005] Hemoglobin extracted from red blood cells has been used as a
blood substitute for oxygen delivery to hypoxic tissues. However,
the use of unmodified cell-free Hb purified from red blood cells
can suffer from several limitations, such as contamination, supply
limitations, an increase in oxygen affinity due to loss of the
cofactor 2,3-disphosphoglycerate (2,3-DPG), and dissociation of Hb
tetramers into .alpha..beta. dimers, which are cleared by renal
filtration and can cause long-term kidney damage.
[0006] The development of improved recombinant hemoglobins can
solve some or all of the above-mentioned problems. However,
existing recombinant hemoglobins can suffer from low oxygen
carrying capacity, and the existing methods for preparing
recombinant hemoglobins are laborious and can suffer from poor
soluble yields of hemoglobin with high percentages of impurities.
For example, protoporphyrin-IX (PPIX), a common byproduct in
conventional recombinant hemoglobin production, can impair the
overall oxygen carrier function of the recombinant hemoglobins.
[0007] Existing methods for preparing recombinant hemoglobins
generally include a heat treatment step to remove impurities, such
as PPIX. However, the incorporation of a heat treatment step into
the preparation method can increase cost and reduce yield of the
protein product. In addition, in order to maintain the stability of
the recombinant hemoglobins during the heat treatment and
downstream processes thereof, CO is required for the recombinant
hemoglobins. However, since CO is undesired in the final product, a
CO removal step is usually required for existing preparation
methods, which also makes the existing methods costly and
time-consuming.
[0008] Therefore, there is a need for improved recombinant
hemoglobins with high oxygen carrying capacity, and there is also a
need for simplified methods for preparing recombinant hemoglobins
with high yield and high purity.
SUMMARY
[0009] The present disclosure generally relates to recombinant
hemoglobins and methods of use and preparation thereof. The
recombinant hemoglobins described herein can have improved
oxygen-carrying capacity. The disclosed recombinant hemoglobins are
useful for treating diseases, such as cancer, stroke, hemorrhagic
shock, acute mountain sickness (AMS), peripheral arterial disease
(PAD) and Parkinson's disease (PD).
[0010] The present disclosure also generally relates to methods of
preparing the recombinant hemoglobins described herein. The
recombinant hemoglobins prepared using the methods described herein
can have reduced amounts of PPIX, little or no HbCO, and an optimal
heme/protein ratio and tetramer/dimer distribution.
[0011] In a first aspect of the present disclosure, there is
provided a recombinant hemoglobin comprising a di-alpha chain and
two beta chains, wherein the di-alpha chain comprises a polypeptide
sequence having at least 98.93% sequence homology with SEQ ID NO:
1, wherein the amino acids at position 1 and position 143 of SEQ ID
NO: 1 must be methionine, the amino acids at position 29 and
position 171 of SEQ ID NO: 1 must be phenylalanine, and the amino
acids at position 58 and position 200 of SEQ ID NO: 1 must be
glutamine.
[0012] In a first embodiment of the first aspect of the present
disclosure, the di-alpha chain comprises a polypeptide sequence
having at least 99.29% sequence homology with SEQ ID NO: 1.
[0013] In a second embodiment of the first aspect of the present
disclosure, the di-alpha chain comprises a polypeptide having at
least 99.64% sequence homology with SEQ ID NO: 1.
[0014] In a third embodiment of the first aspect of the present
disclosure, the di-alpha chain is a polypeptide sequence having SEQ
ID NO: 1.
[0015] In a fourth embodiment of the first aspect of the present
disclosure, each of the two beta chains comprises a polypeptide
sequence having at least 97.94% sequence homology with SEQ ID NO:
2, wherein the amino acid at position 1 must be methionine.
[0016] In a fifth embodiment of the first aspect of the present
disclosure, each of the two beta chains is a polypeptide sequence
having SEQ ID NO: 2.
[0017] In a sixth embodiment of the first aspect of the present
disclosure, each of the two beta chains comprises a polypeptide
sequence having at least 97.94% sequence homology with SEQ ID NO:
3, wherein the amino acid at position 1 must be methionine, the
amino acid at position 82 must be aspartic acid, and the amino acid
at position 108 must be lysine.
[0018] In a seventh embodiment of the first aspect of the present
disclosure, each of the two beta chains comprises a polypeptide
sequence having at least 98.63% sequence homology with SEQ ID NO:
3.
[0019] In an eighth embodiment of the first aspect of the present
disclosure, each of the two beta chains comprises a polypeptide
sequence having at least 99.31% sequence homology with SEQ ID NO:
3.
[0020] In a ninth embodiment of the first aspect of the present
disclosure, each of the two beta chains comprises a polypeptide
sequence of SEQ ID NO: 3.
[0021] In a second aspect of the present disclosure, there is
provided a pharmaceutical composition comprising the
above-mentioned recombinant hemoglobin and at least one
pharmaceutically acceptable carrier.
[0022] In a third aspect of the present disclosure, there is
provided a method of treating an oxygen deficiency related disease
in a subject in need thereof comprising the step of administering a
therapeutically effective amount of the pharmaceutical composition
to the subject.
[0023] In a first embodiment of the third aspect of the present
disclosure, the oxygen deficiency related disease comprises cancer,
stroke, hemorrhagic shock, acute mountain sickness (AMS),
peripheral arterial disease (PAD) or Parkinson's disease (PD).
[0024] In a fourth aspect of the present disclosure, there is
provided a method of producing the recombinant hemoglobin,
comprising the steps of: [0025] (a) providing a host cell
comprising a polynucleotide encoding the di-alpha chain and a
polynucleotide encoding the beta chain; and [0026] (b) inducing the
host cell containing the polynucleotide encoding the di-alpha chain
and the polynucleotide encoding the beta chain to express
recombinant hemoglobin thereby producing the recombinant
hemoglobin.
[0027] In a first embodiment of the fourth aspect of the present
disclosure, the polynucleotide encoding the di-alpha chain and the
polynucleotide encoding the beta chain comprise a polynucleotide
sequence of SEQ ID NO: 4 and a polynucleotide sequence of SEQ ID
NO: 5, respectively.
[0028] In a second embodiment of the fourth aspect of the present
disclosure, the polynucleotide encoding the di-alpha chain and the
polynucleotide encoding the beta chain comprises a polynucleotide
sequence of SEQ ID NO: 4 and a polynucleotide sequence of SEQ ID
NO: 6, respectively.
[0029] In a third embodiment of the fourth aspect of the present
disclosure, the host cell is selected from the group consisting of
JM109 E. coli bacterial strain with lambda DE3, BL21-AI E. coli
bacterial strain without lambda DE3, and SHuffle E. coli bacterial
strain without lambda DE3.
[0030] In a fourth embodiment of the fourth aspect of the present
disclosure, the host cell further comprises a polynucleotide
encoding HemH.
[0031] In a fifth embodiment of the fourth aspect of the present
disclosure, the host cell further comprises a polynucleotide
encoding a Heme-transporter.
[0032] In a sixth embodiment of the fourth aspect of the present
disclosure, the method further comprises the steps of: [0033] (a)
rupturing the host cell after the step of inducing the host cell to
express recombinant hemoglobin to obtain a solution comprising the
recombinant hemoglobin; and [0034] (b) purifying the recombinant
hemoglobin to obtain a purified recombinant hemoglobin.
[0035] In a firth aspect of the present disclosure, there is
provided a system for producing the recombinant hemoglobin of the
first aspect of the present disclosure, the system comprising: an
Escherichia coli or non-Escherichia coli host cell comprising: a
polynucleotide encoding HemH; a polynucleotide encoding a
Heme-transporter; a polynucleotide encoding a di-alpha chain; and a
polynucleotide encoding a beta chain, wherein the di-alpha chain
comprises a polypeptide sequence having at least 98.93% sequence
homology with SEQ ID NO: 1, wherein the amino acids at position 1
and position 143 of SEQ ID NO: 1 must be methionine, the amino
acids at position 29 and position 171 of SEQ ID NO: 1 must be
phenylalanine, and the amino acids at position 58 and position 200
of SEQ ID NO: 1 must be glutamine; and the beta chain comprises a
polypeptide sequence having at least 99.31% sequence homology with
SEQ ID NO: 2, wherein the amino acid at position 1 must be
methionine; or a polypeptide sequence having at least 97.94%
sequence homology with SEQ ID NO: 3, wherein the amino acid at
position 1 must be methionine, the amino acid at position 82 must
be aspartic acid, and the amino acid at position 108 must be
lysine.
[0036] Those skilled in the art will appreciate that the disclosure
described herein is susceptible to variations and modifications
other than those specifically described.
[0037] Other aspects and advantages of the disclosure will be
apparent to those skilled in the art from a review of the ensuing
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows the polypeptide sequences for di-alpha chain
SEQ ID NO: 1 and SEQ ID NO: 11 and beta chain SEQ ID NO: 2, SEQ ID
NO: 3, and SEQ ID NO: 12 according to certain embodiments described
herein.
[0039] FIG. 2A shows the plasmid maps, where pACYCDuet-CT is the
vector carrying the ChuA gene and the gene for expressing TonB, and
pET-HemHm1-TB is a general plasmid that carries the sequence for
expressing HemH mutant and TB, where TB can be the sequence for
expressing TBN, TBM1, or TBM9.
[0040] FIG. 2B shows the SDS-PAGE analysis of TBN, TBM1 and TBM9
expression in different cell strains by the plasmid in FIG. 2A,
wherein #83 is the result for TBM1 expressed in SHuffle E. coli
bacterial strain without lambda DE3, #84 is the result for TBM9
expressed in SHuffle E. coli bacterial strain without lambda DE3,
#85 is the result for TBM1 expressed in JM109 T7CT CRISPR/Cas9 E.
coli bacterial strain without lambda DE3, #86 is the result for
TBM9 expressed in JM109 T7CT CRISPR/Cas9 E. coli bacterial strain
without lambda DE3, and #43 is the result for TBN expressed in
JM109 E. coli bacterial strain with lambda DE3.
[0041] FIG. 2C shows the SDS-PAGE analysis of TBN, TBM1 and TBM9
expression in different cell strains by the plasmid in FIG. 2A,
wherein #83 is the result for TBM1 expressed in SHuffle E. coli
bacterial strain without lambda DE3, #84 is the result for TBM9
expressed in SHuffle E. coli bacterial strain without lambda DE3,
#85 is the result for TBM1 expressed in JM109 T7CT CRISPR/Cas9 E.
coli bacterial strain without lambda DE3, #86 is the result for
TBM9 expressed in JM109 T7CT CRISPR/Cas9 E. coli bacterial strain
without lambda DE3, and #43 is the result for TBN expressed in
JM109 E. coli bacterial strain with lambda DE3.
[0042] FIG. 3A shows the illustration of the liver oxygen level
measurement in a mouse model.
[0043] FIG. 3B shows the change of the liver oxygen level of the
mouse model over 180 min after the administration of TBN, TBM1,
TBM9, YQ (bovine fumaryl crosslinked Hb), and RA-buffer as a
control.
[0044] FIG. 4A shows a ultra-high performance liquid chromatography
(UPLC) chromatogram showing the heme and PPIX levels of purified
protein expressed using the previous plasmid comprising the
nucleotide sequences of TBN (FIG. 4A).
[0045] FIG. 4B shows a UPLC chromatogram showing the heme and PPIX
level of purified protein expressed using the new improved plasmid
comprising nucleotide sequences of HemH mutant, Heme transporter
system (ChuA and TonB), and TBN (FIG. 4B).
[0046] FIG. 5A shows the oxygen equilibrium curve for different
recombinant human hemoglobins (TBN, TBM1, and TBM9) according to
certain embodiments described herein.
[0047] FIG. 5B shows a table of the p50 value of different
recombinant human hemoglobins (TBN, TBM1, and TBM9) with and
without 2,3-DPG according to certain embodiments described
herein.
[0048] FIG. 5C shows an oxygen equilibrium curve of TBM1 with and
without 2,3-DPG according to certain embodiments described
herein.
[0049] FIG. 6 shows a purification flow-chart according to certain
embodiments described herein.
[0050] FIG. 7 shows the HemH mutant polynucleotide SEQ ID NO: 8
(top) and HemH mutant protein SEQ ID NO: 13 (bottom) according to
certain embodiments described herein.
[0051] FIG. 8 shows the ChuA polynucleotide SEQ ID NO: 9 (top) and
ChuA protein SEQ ID NO: 14 (bottom) according to certain
embodiments described herein.
[0052] FIG. 9 shows the TonB polynucleotide SEQ ID NO: 10 (top) and
TonB protein SEQ ID NO: 15 (bottom) according to certain
embodiments described herein.
[0053] FIG. 10 shows a graph (top) and table (bottom) showing the
azide-induced oxidation rates (k.sub.az) values of TBN, TBM1, and
TBM9.
[0054] FIG. 11 shows MTT assay results for recombinant human
hemoglobins TBM1 and TBM9 inhibiting 4T1 breast cancer cell
proliferation in vitro.
[0055] FIG. 12A show the recombinant human hemoglobins (TBM1)
suppressing 4T1 breast cancer cell growth in vivo.
[0056] FIG. 12B shows a bar graph showing the recombinant human
hemoglobins (TBM1) suppressing 4T1 breast cancer cell growth in
vivo.
[0057] FIG. 13 shows the results of a murine PAD model results
using saline and recombinant human hemoglobin (TBM1).
[0058] FIG. 14 shows body weight results after treating with
recombinant human hemoglobin (TBM1) in Balb/c and ICR mice.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present disclosure relates to recombinant hemoglobins
having high oxygen carrying capacity. The recombinant hemoglobins
described herein comprise a di-alpha chain and two beta chains.
Definition of Terms
[0060] The definitions of terms used herein are meant to
incorporate the present state-of-the-art definitions recognized for
each term in the field of biotechnology. Where appropriate,
exemplification is provided. The definitions apply to the terms as
they are used throughout this specification, unless otherwise
limited in specific instances, either individually or as part of a
larger group.
[0061] The term "recombinant hemoglobin(s)" as used herein
indicates a hemoglobin molecule and/or its variant with a molecular
size of at least approximately 65 kDa and is synthesized by any
standard molecular biology techniques rather than being isolated or
purified from any animal or human source.
[0062] The term "protein" or "polypeptide" as used herein indicates
an organic polymer composed of two or more amino acid monomers
and/or analogs thereof The term "polypeptide" includes amino acid
polymers of any length including full length proteins and peptides,
as well as analogs and fragments thereof. A polypeptide of three or
more amino acids is also called an oligopeptide. As used herein,
the term "amino acid", "amino acid monomer", or "amino acid
residue" refers to any of the twenty naturally occurring amino
acids including synthetic amino acids with unnatural side chains
and including both D and L optical isomers. The terms "amino acid
analog" and "analog" that are used interchangeably refer to an
amino acid in which one or more individual atoms have been
replaced, either with a different atom, isotope, or with a
different functional group but is otherwise identical to its
natural amino acid analog and have similar chemical and/or physical
properties to its natural amino acid analog.
[0063] As used herein, the term "variant" refers to a polypeptide
or polynucleotide sequence differing from a reference polypeptide
or polynucleotide sequence, but retaining essential properties
thereof. Generally, variants are overall closely similar, and, in
many regions, identical to the reference polypeptide or
polynucleotide sequence.
[0064] A variant can, for example, comprise the amino acid sequence
of the parent polypeptide sequence with at least one conservative
amino acid substitution. Alternatively or additionally, the variant
can comprise the amino acid sequence of the parent polypeptide
sequence with at least one non-conservative amino acid
substitution. In this case, it is preferable for the
non-conservative amino acid substitution to not interfere with or
inhibit the biological activity of the functional variant. The
non-conservative amino acid substitution may enhance the biological
activity of the variant, such that the biological activity of the
variant is increased as compared to the parent polypeptide.
[0065] The term "amino acid modification" as used herein indicates
amino acid insertion, substitution, or deletion, etc. Amino acid
substitutions of the described polypeptides can be conservative
amino acid substitutions. Conservative amino acid substitutions are
known in the art, and include amino acid substitutions in which one
amino acid having certain physical and/or chemical properties is
exchanged for another amino acid that has the same or similar
chemical or physical properties. For instance, the conservative
amino acid substitution can be an acidic/negatively charged polar
amino acid substituted for another acidic/negatively charged polar
amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side
chain substituted for another amino acid with a nonpolar side chain
(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val,
etc.), a basic/positively charged polar amino acid substituted for
another basic/positively charged polar amino acid (e.g. Lys, His,
Arg, etc.), an uncharged amino acid with a polar side chain
substituted for another uncharged amino acid with a polar side
chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a
beta-branched side-chain substituted for another amino acid with a
beta-branched side-chain (e.g., Ile, Thr, and Val), an amino acid
with an aromatic side-chain substituted for another amino acid with
an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.
[0066] The term "nucleotide modifications" as used herein refers to
nucleotide insertion, substitution, deletion, etc.
[0067] The term "percentage sequence homology", when used in
reference to a polypeptide or polynucleotide sequence, refers to
comparisons among polynucleotides and polypeptides, and are
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide or
polypeptide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions for
the longer sequence in the window of comparison and multiplying the
result by 100 to yield the percentage of sequence homology.
Homology is evaluated using any of the variety of sequence
comparison algorithms and programs known in the art. Such
algorithms and programs include, but are by no means limited to,
TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,
1988, Proc. Natl. Acad. Sci. USA 85(8): 2444-2448; Altschul et al.,
1990, J. Mol. Biol. 215(3): 403-410; Thompson et al., 1994, Nucleic
Acids Res. 22(2): 4673-4680; Higgins et al. 1996, Methods Enzymol.
266: 383-402; Altschul et al., 1990, J. Mol. Biol. 215(3): 403-410;
Altschul et al., 1993, Nature Genetics 3: 266-272). In certain
embodiments, protein and nucleic acid sequence homologies are
evaluated using the Basic Local Alignment Search Tool ("BLAST")
which is well known in the art (see, e.g., Karlin and Altschul,
1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al.,
1990, J. Mol. Biol. 215: 403-410; Altschul et al., 1993, Nature
Genetics 3: 266-272; Altschul et al., 1997, Nuc. Acids Res. 25:
3389-3402).
[0068] As used herein, the terms "treat", "treating", "treatment",
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated, although not
precluded, treating a disorder or condition does not require that
the disorder, condition, or symptoms associated therewith be
completely eliminated. In certain embodiments, treatment includes
prevention of a disorder or condition, and/or symptoms associated
therewith.
[0069] As used herein the term "stroke" indicates a medical
condition in which poor blood flow to the brain results in cell
death. It includes but not limited to ischemic stroke and
hemorrhagic stroke.
[0070] As used herein, the term "hemorrhagic shock" refers to a
condition of reduced tissue perfusion, resulting in the inadequate
delivery of oxygen and nutrients that are necessary for cellular
function.
[0071] The term "acute mountain sickness" as used herein refers to
a condition developed when exposed to high altitudes having low
levels of oxygen and decreased air pressure.
[0072] The term "ischemic disease" or "ischemia" as used herein
refers to diseases and/or conditions characterized by reduced
oxygenation to any tissue in the body such as, but not limited to,
ischemic heart disease, transient ischemic attack, cardiac
ischemia, stroke, reperfusion injury, bowel ischemia, intestinal
ischemia, peripheral artery disease, critical limb ischemia,
mesenteric ischemia, brain ischemia, leg ischemia, myocardial
infarction, peripheral vascular disease, coronary artery disease,
angina, wound healing, renal artery disease, diabetic ulcer
healing, congestive heart failure, and hepatic ischemia. Ischemia
can be caused by a number of conditions including, but not limited
to, anemia, stroke and atherosclerosis. Multiple diseases result
from ischemia including, for example, cerebrovascular ischemia,
renal ischemia, pulmonary ischemia, limb ischemia, myocardial
ischemia, and ischemic cardiomyopathy.
[0073] The term "peripheral artery disease" or "PAD" as used herein
refers to a condition of narrowing of the peripheral arteries, such
as those serving the legs, stomach, arms and/or head. PAD includes
a wide range of vascular diseases caused by atherosclerotic,
thromboembolic, and inflammatory processes that alter the structure
and function of the arteries. The most common cause of PAD is
atherosclerosis.
[0074] The term "p50 value" as used herein indicates the oxygen
tension at which hemoglobin is 50% saturated. Values of p50 are
negatively correlated with substrate affinity; lower values
correspond to higher affinity and vice versa.
[0075] The term "oxygen carrying capacity" as used herein indicates
the capability of a composition to carry oxygen. The capability to
carry oxygen comprises but not limited to two aspects: compositions
containing recombinant hemoglobins with lower oxygen affinity are
used when rapid oxygenation is desired in cases of tissue hypoxia
resulting from extensive blood loss (e.g., hemorrhagic shock).
Lower oxygen affinity means that the material can "offload" oxygen
to a target more easily than a material with a higher oxygen
affinity. Compositions with higher oxygen affinity are useful as
oxygenation adjunct therapies in cancer treatment where a slower
delivery rate of oxygen is desired in that case. "Improved oxygen
carrying capacity" means the level of oxygen carried by a
composition is increased regardless of rapid oxygenation or low
delivery of oxygen.
[0076] The terms "co-transform", "co-transforming",
"co-transformed", and the like refer to a process that transfers
more than one plasmids or vectors bearing exogenous DNA into the
host cell at the same time. The host cells generally include but
are not limited to bacterial, yeast, insect, mammalian, and plant
cells. Any other reasonable host cells for accepting plasmids or
vectors bearing exogenous DNA are also within the contemplation of
the present disclosure. The methods for transformation of a DNA
construct into a host cell include but not limited to chemical
transformation, electroporation or particle bombardment. Any other
reasonable methods for transforming a DNA construct into a host
cell are also within the contemplation of the present disclosure.
As used herein, the terms "plasmid", "expression plasmid",
"vector", "DNA construct" and the like are used interchangeably
herein to refer to a genetic structure that carries one or more
exogenous DNA sequences of interest, as well as other functional
DNA segments and/or sites, such as promoter segment, restriction
site, 5' primer site, 3' primer site, origin of replication
segment, antibiotic resistance gene segment, selectable marker
segment, etc. These genetic structures are typically, but not
always, circular DNA molecules, which are physically separated from
a chromosomal DNA and can replicate independently. The terms
"exogenous DNA", "exogenous DNA sequences of interest" and the like
as used herein refer to deoxyribonucleic acid that originates
outside of the host cells, including but not limited to the genes
encoding particular proteins of interest or subunits thereof. The
term "recombinant hemoglobin expression plasmid" as used herein can
refer to a plasmid that carries a polynucleotide sequence encoding
one di-alpha chain, a plasmid that carries a polynucleotide
sequence encoding one or two beta chains, or a plasmid that carries
a polynucleotide sequence encoding a di-alpha chain and a beta
chain.
[0077] The term "HemH" as used herein refers to any enzyme, such as
ferrochelatase, that catalyzes the production of heme. The term
"Heme-transporter" as used herein refer proteins that facilitate
the uptake of heme and/or iron, and such proteins include, but are
not limited to, ChuA, TonB, heme carrier protein 1 (HCP1), divalent
metal transporter 1 (DMT1), mucolipin-1 (also known as TRPML1),
HRG1, and hemopexin.
[0078] The term "HemH plasmid" as used herein refers to a plasmid
that carries a polynucleotide sequence encoding any enzyme, such as
ferrochelatase, that catalyzes the production of heme, and the
polynucleotide may include one or more nucleotide modifications.
The term "Heme-transporter plasmid (s)" as used herein refer to one
or more plasmids that carry one or more polynucleotide sequences
encoding one or more proteins that facilitate the uptake of heme
and/or iron, and such proteins include, but are not limited to,
ChuA, TonB, heme carrier protein 1 (HCP1), divalent metal
transporter 1 (DMT1), mucolipin-1 (also known as TRPML1), HRG1, and
hemopexin.
[0079] The di-alpha chain can comprise a polypeptide sequence
having at least 98.93% sequence homology with SEQ ID NO: 1.
Polypeptides having at least 98.93% sequence homology to SEQ ID
NO:1 can refer to polypeptides having at most three amino acid
modifications, i.e. zero, one, two, or three amino acid
modifications with respect to the SEQ ID NO: 1. In certain
embodiments, the di-alpha chain comprises a polypeptide sequence
having at least 99.29% sequence homology with the SEQ ID NO: 1.
Polypeptides having at least 99.29% sequence homology can have at
most two amino acid modifications, i.e. zero, one, or two amino
acid modifications with respect to the SEQ ID NO: 1. In certain
embodiments, the di-alpha chain comprises a polypeptide sequence
having at least 99.64% sequence homology with the SEQ ID NO: 1.
Polypeptides having at least 99.64% sequence homology can have at
most one amino acid modifications, i.e. zero or one amino acid
modification with respect to the SEQ ID NO: 1. In certain
embodiments, the di-alpha chain comprises a polypeptide sequence of
SEQ ID NO: 1. In certain embodiments, the di-alpha chain consists
of a polypeptide sequence of SEQ ID NO: 1. The one, two, or three
amino acid modifications can occur at any amino acid present in SEQ
ID NO: 1, except the positions 1, 29, 58, 143, 171, and 200 of SEQ
ID NO: 1, in which position 1 and position 143 of SEQ ID NO: 1 must
be methionine, the amino acids at position 29 and position 171 of
SEQ ID NO: 1 must be phenylalanine, and the amino acids at position
58 and position 200 of SEQ ID NO: 1 must be glutamine.
[0080] The polypeptide sequence of the di-alpha chain can contain a
linker that connects the N-terminal of a first alpha subunit with
the C-terminal of a second alpha subunit or the N-terminal of a
first alpha subunit is directly connected to the C-terminal of a
second alpha subunit. In instances where a linker is presented in
the di-alpha chain, the linker can be one or more amino acid
residues selected from the group consisting of glycine and serine.
In certain embodiments, the linker is (Gly-Ser).sub.n,
(Gly-Gly-Gly-Ser).sub.n, (Gly-Gly-Ser-Gly).sub.n,
(Gly-Gly-Gly-Gly-Ser).sub.n, (Gly-Gly-Ser)n, (Gly-Ser).sub.n or
Gly.sub.n, wherein n is 1-10. In certain embodiments, the linker is
Gly.sub.n linker, wherein n is 1-4. In certain embodiments, the
linker is Gly.
[0081] Each of the two beta chains can comprise a polypeptide
sequence having at least 97.94% sequence homology with SEQ ID NO:
2. Polypeptides having at least 97.94% sequence homology can have
at most three amino acid modifications, i.e. zero, one, two, or
three amino acid modifications with respect to the SEQ ID NO: 2. In
certain embodiments, each of the two beta chains comprises a
polypeptide sequence having at least 98.63% sequence homology with
the SEQ ID NO: 2. Polypeptides having at least 98.63% sequence
homology can have at most two amino acid modifications, i.e. zero,
one, or two amino acid modifications with respect to the SEQ ID NO:
2. In certain embodiments, each of the two beta chains comprises a
polypeptide sequence having at least 99.31% sequence homology with
the SEQ ID NO: 2. Polypeptides having at least 99.31% sequence
homology can have at most one amino acid modifications, i.e. zero
or one amino acid modification with respect to the SEQ ID NO: 2. In
certain embodiments, each of the two beta chains comprises a
polypeptide sequence of SEQ ID NO: 2. In certain embodiments, the
one, two, or three amino acid modifications can occur at any amino
acid present in SEQ ID NO: 2, except the position 1 of the SEQ ID
NO: 2, which must be methionine.
[0082] In certain embodiments, each of the two beta chains can
comprise a polypeptide sequence having at least 97.94% sequence
homology with SEQ ID NO: 3. Polypeptides having at least 97.94%
sequence homology can refer to polypeptides having at most three
amino acid modifications, i.e. zero, one, two, or three amino acid
modifications with respect to the SEQ ID NO: 3. In certain
embodiments, each of the two beta chains comprises a polypeptide
sequence having at least 98.63% sequence homology with the SEQ ID
NO: 3. Polypeptides having at least 98.63% sequence homology can
have at most two amino acid modifications, i.e. zero, one, or two
amino acid modifications with respect to the SEQ ID NO: 3. In
certain embodiments, each of the two beta chains comprises a
polypeptide sequence having at least 99.31% sequence homology with
the SEQ ID NO: 3. Polypeptides having at least 99.31% sequence
homology can have at most one amino acid modifications, i.e. zero
or one amino acid modification with respect to the SEQ ID NO: 3. In
certain embodiments, each of the two beta chains comprises a
polypeptide sequence of SEQ ID NO: 3. The one, two, or three amino
acid modifications can occur at any amino acid present in SEQ ID
NO: 3, except the positions 1, 82, and 108 of the SEQ ID NO: 3, in
which position 1 of the SEQ ID NO: 3 must be methionine, the
position 82 of the SEQ ID NO: 3 must be aspartic acid, and the
position 108 of the SEQ ID NO: 3 must be lysine.
[0083] Di-alpha chain having a polypeptide sequence of SEQ ID NO:
11 and two of the beta chains having a polypeptide sequence of SEQ
ID NO: 12 form the recombinant hemoglobin TBN. Di-alpha chain
having a polypeptide sequence of SEQ ID NO: 1 and two of the beta
chains having a polypeptide sequence of SEQ ID NO: 2 form the
recombinant hemoglobin TBM1. Di-alpha chain having a polypeptide
sequence of SEQ ID NO: 1 and two of the beta chains having a
polypeptide sequence of SEQ ID NO: 3 form the recombinant
hemoglobin TBM9.
[0084] PPIX concentrations of TBN, TBM1, and TBM9 prepared under
identical conditions are listed in Table 1. The p50 level, tetramer
purity, met-hemoglobin (Met-Hb)% and heme/protein ratio are shown
in Table 2. The methods for preparing the recombinant hemoglobins
described herein are simplified and cost effective. Without wishing
to be bound by theory, it is believed that the mutations of the
di-alpha chain and beta chain lead to structural changes of the
recombinant hemoglobins, which affects the kinetics of the
incorporation of heme and PPIX, and ultimately changes the
percentage of PPIX in the purified recombinant hemoglobins.
TABLE-US-00001 TABLE 1 PPIX % in different purified recombinant
hemoglobins Recombinant hemoglobin *PPIX % TBN 1.05 TBM1 0.11 TBM9
0.08 (*The PPIX % value is the average from five batches)
TABLE-US-00002 TABLE 2 Properties of purified recombinant
hemoglobins Measurement TBN TBM1 TBM9 p50 level (mmHg) Raw: 14.86
Raw: 23.87 Raw: 35.80 (with 2,3-DPG) Adair: 14.55 Adair: 25.38
Adair: 40.82 Purity (Tetramer) (%) 97.5 100 100 Met-Hb (%) 6.6 3.8
3.2 (by nanodrop method) Heme/Protein ratio 3.59 3.72 3.94
[0085] The present disclosure also relates to methods for
expressing, fermenting, and purifying the recombinant hemoglobins
described herein. In certain embodiments, the method comprises
co-expressing the plasmids encoding the recombinant hemoglobins
described herein, a ferrochelatase (HemH) plasmid, and a
Heme-transporter plasmid in the host cells. Different host cells
listed in Table 3 were tested, and among the host cells, JM109
(DE3) (with lambda DE3) (i.e. JM109 E. coli bacterial strain with
lambda DE3), BL21-T7 or AI E. coli bacterial strain (without lambda
DE3) and SHuffle.RTM. T7 Competent E. coli (without lambda DE3)
(i.e. SHuffle E. coli bacterial strain without lambda DE3) have the
best yield and quality. These four host cells are easily scalable,
can express the plasmids transformed therewithin quickly, are very
cheap, require simple culture conditions, and have established
regulatory track record. The HemH plasmid can be wildtype or
contain certain mutations. The Heme-transporter plasmid can be
constructed by inserting the E. coli heme-utilization gene (ChuA)
into a vector. The ChuA gene encodes a 69 kDa outer membrane
protein, which facilitates the uptake of heme in E. coli. The
uptake of heme by ChuA is dependent on an inner membrane protein
named TonB. Therefore, the methods described herein preferably
comprise a step of co-transforming the recombinant hemoglobin
plasmids, the HemH plasmid, the ChuA plasmid, and the TonB plasmid
into the selected host cells, i.e. JM109 E. coli bacterial strain
with lambda DE3, BL21-T7 or AI E. coli bacterial strain without
lambda DE3 and SHuffle E. coli bacterial strain without lambda DE3.
Alternatively, the method can also co-transform the ChuA plasmid
alone together with the recombinant hemoglobin plasmids and the
HemH plasmid into the selected host cells.
[0086] The gene encoding the ferrochelatase (HemH) and the
Heme-transporter can also be inserted into genome of E. coli
bacterial strain by CRISPR/Cas9 genome editing method to
endogenously express ferrochelatase (HemH) and the
Heme-transporter. By this method, BL21-T7 and Jm109-T7 E. coli
bacterial strain were modified to BL21-T7-CT(CRISPR/Cas9) and
Jm109-T7-CT(CRISPR/Cas9) bacterial strains to express recombinant
hemoglobin proteins.
TABLE-US-00003 TABLE 3 Different host cells No. Strain lambda DE3 1
JM109 (DE3) Y 2 BL21 (DE3) Y 3 BLR(DE3) Y 4 HMS174(DE3) Y 5
NovaBlue(DE3) Y 6 Tuner(DE3) Y 7 Origami 2 .TM.(DE3) Y 8 Origami
B(DE3) Y 9 Rosetta .TM. 2(DE3) Y 10 Rosetta-gami(DE3) Y 11
Rosetta-gami B(DE3) Y 12 Rosetta Blue(DE3) Y 13 BL21 Star (DE3) Y
14 BL21 Super Star (DE3) Y 15 T7 Express Competent E. coli N (High
Efficiency) 16 T7 Express lysY Competent E. coli N (High
Efficiency) 17 T7 Express lysY/Iq Competent E. coli N (High
Efficiency) 18 ScarabXpress T7lac N 19 SHuffle .RTM. T7 Competent
E. coli N 20 BL21-AI .TM. One Shot .TM. Chemically N Competent E.
coli 21 BL21-T7 N 22 BL21-T7-CT(CRISPR/Cas9) N 23
JM109-T7-CT(CRISPR/Cas9) N (Y: Yes, N: No)
[0087] The method further comprises a purification step that
advantageously does not require a heat step, and does not use
carbon monoxide. The steps for purification are shown in FIG. 6.
The recombinant hemoglobins prepared using the methods described
herein can have reduced amounts of protoporphyrin-IX (PPIX), e.g.,
less than 1% (by weight), shown in Table 1. In certain embodiments,
the recombinant hemoglobins prepared using the methods described
herein have between 0.08% and 1%, 0.11% and 1%, 0.08% and 0.20%,
0.08% and 0.15%, or 0.08% and 0.11% (by weight) of PPIX. As such,
the methods described herein are simpler as compared with existing
methods for recombinant hemoglobin preparation, and can result in
higher yield of soluble recombinant hemoglobin with reduced levels
of impurities.
[0088] The present disclosure further relates to pharmaceutical
compositions comprising the recombinant hemoglobins described
herein and at least one pharmaceutically acceptable carrier. In
certain embodiments, the pharmaceutical composition comprises other
excipients, such as amino acids and sugar.
[0089] The present disclosure further relates to a polynucleotide
sequence encoding a di-alpha chain as described herein. In certain
embodiments, the polynucleotide sequence encodes a di-alpha chain
comprising a polypeptide sequence having at least 98.93% sequence
homology with SEQ ID NO: 1, wherein the amino acids at position 1
and position 143 of SEQ ID NO: 1 must be methionine, the amino
acids at position 29 and position 171 of SEQ ID NO: 1 must be
phenylalanine, and the amino acids at position 58 and position 200
of SEQ ID NO: 1 must be glutamine. In certain embodiments, the
polynucleotide sequence encodes a di-alpha chain comprising a
polypeptide sequence having at least 99.29% sequence homology with
the SEQ ID NO: 1, wherein the amino acids at position 1 and
position 143 of SEQ ID NO: 1 must be methionine, the amino acids at
position 29 and position 171 of SEQ ID NO: 1 must be phenylalanine,
and the amino acids at position 58 and position 200 of SEQ ID NO: 1
must be glutamine. In certain embodiments, the polynucleotide
sequence encodes a di-alpha chain comprising a polypeptide sequence
having at least 99.64% sequence homology with the SEQ ID NO: 1,
wherein the amino acids at position 1 and position 143 of SEQ ID
NO: 1 must be methionine, the amino acids at position 29 and
position 171 of SEQ ID NO: 1 must be phenylalanine, and the amino
acids at position 58 and position 200 of SEQ ID NO: 1 must be
glutamine. In certain embodiments, the polynucleotide sequence
encodes a di-alpha chain comprising a polypeptide sequence of SEQ
ID NO: 1. In certain embodiments, the polynucleotide sequence
encodes a di-alpha chain consisting of a polypeptide sequence of
SEQ ID NO: 1.
[0090] The present disclosure further relates to a polynucleotide
sequence encoding a beta chain as described herein. In certain
embodiments, the polynucleotide sequence encodes a beta chain
comprising a polypeptide sequence having at least 97.94% sequence
homology with SEQ ID NO: 2. In certain embodiments, the
polynucleotide sequence encodes a beta chain comprising a
polypeptide sequence having at least 98.63% sequence homology with
the SEQ ID NO: 2. In certain embodiments, the polynucleotide
sequence encodes a beta chain comprising a polypeptide sequence
having at least 99.31% sequence homology with the SEQ ID NO: 2. In
certain embodiments, the polynucleotide sequence encodes a beta
chain comprising a polypeptide sequence comprising a polypeptide
sequence of SEQ ID NO: 2. In certain embodiments, the
polynucleotide sequence encodes a beta chain comprising a
polypeptide sequence consisting of a polypeptide sequence of SEQ ID
NO: 2.
[0091] In certain embodiments, the polynucleotide sequence encodes
a beta chain comprising a polypeptide sequence having at least
97.94% sequence homology with SEQ ID NO: 3 In certain embodiments,
the polynucleotide sequence encodes a beta chain comprising a
polypeptide sequence having at least 98.63% sequence homology with
the SEQ ID NO: 3. In certain embodiments, the polynucleotide
sequence encodes a beta chain comprising a polypeptide sequence
having at least 99.31% sequence homology with the SEQ ID NO: 3. In
certain embodiments, the polynucleotide sequence encodes a beta
chain comprising a polypeptide sequence comprising a polypeptide
sequence of SEQ ID NO: 3. In certain embodiments, the acid
modifications can occur at any amino acid present in SEQ ID NO: 3,
except the positions 1, 82, and 108 of the SEQ ID NO: 3, in which
position 1 of the SEQ ID NO: 3 must be methionine, the position 82
of the SEQ ID NO: 3 must be aspartic acid, and the position 108 of
the SEQ ID NO: 3 must be lysine.
[0092] In certain embodiments, the polynucleotide sequence encodes
a di-alpha chain as described herein and a beta chain as described
herein. In certain embodiments, the di-alpha chain comprises a
polypeptide sequence having at least 98.93% sequence homology with
SEQ ID NO: 1, wherein the amino acids at position 1 and position
143 of SEQ ID NO: 1 must be methionine, the amino acids at position
29 and position 171 of SEQ ID NO: 1 must be phenylalanine, and the
amino acids at position 58 and position 200 of SEQ ID NO: 1 must be
glutamine; and the two beta chains comprises a polypeptide sequence
having at least 99.31% sequence homology with SEQ ID NO: 2, wherein
the amino acid at position 1 must be methionine; or a polypeptide
sequence having at least 97.94% sequence homology with SEQ ID NO:
3, wherein the amino acid at position 1 must be methionine, the
amino acid at position 82 must be aspartic acid, and the amino acid
at position 108 must be lysine.
[0093] The polynucleotides sequences described herein can be
introduced into a desired cell using one or more vectors. A vector
is a typically includes additional sequences, including sequences
that direct propagation of the vector in the cell or insertion of
part of the vector into the cell's genome, and a gene which allows
an individual to screen for the presence of the vector. Common
examples of vectors include plasmids, artificial chromosomes,
viruses, and linear polynucleotide fragments which are designed to
insert into a cell's genome. Vectors are well known tools to a
person of ordinary skill in the art, and a person of ordinary skill
in the art can easily find appropriate vectors for a particular
organism in the literature or in biobanks, such as ATCC. In certain
embodiments, provided herein is a vector comprising a
polynucleotide sequence encoding at least one of a di-alpha chain
described herein and a beta chain as described herein. In certain
embodiments, the vector is a plasmid, artificial chromosomes,
viruses, or a linear polynucleotide fragment, which is designed to
insert into a cell's genome.
[0094] The present disclosure further relates to a system for
producing a recombinant hemoglobin as described herein, the system
comprising: an Escherichia coli or non-Escherichia coli host cell
comprising: a polynucleotide encoding HemH; a polynucleotide
encoding Heme-transporter; a polynucleotide encoding a di-alpha
chain; and a polynucleotide encoding a beta chain; wherein the
di-alpha chain comprises a polypeptide sequence having at least
98.93% sequence homology with SEQ ID NO: 1, wherein the amino acids
at position 1 and position 143 of SEQ ID NO: 1 must be methionine,
the amino acids at position 29 and position 171 of SEQ ID NO: 1
must be phenylalanine, and the amino acids at position 58 and
position 200 of SEQ ID NO: 1 must be glutamine; and the beta chain
comprises a polypeptide sequence having at least 99.31% sequence
homology with SEQ ID NO: 2, wherein the amino acid at position 1
must be methionine; or a polypeptide sequence having at least
97.94% sequence homology with SEQ ID NO: 3, wherein the amino acid
at position 1 must be methionine, the amino acid at position 82
must be aspartic acid, and the amino acid at position 108 must be
lysine. In certain embodiments, the polynucleotide encoding HemH;
the polynucleotide encoding Heme-transporter; the polynucleotide
encoding the di-alpha chain; and the polynucleotide encoding the
beta chain are present in one or more vectors selected from the
group consisting of a plasmid, artificial chromosomes, viruses, and
linear polynucleotide fragments which are designed to insert into a
cell's genome. In certain embodiments, the polynucleotide encoding
HemH; the polynucleotide encoding Heme-transporter; the
polynucleotide encoding the di-alpha chain; and the polynucleotide
encoding the beta chain are inserted into the host cell's genome
using CRISPR/CAS 9. In certain embodiments, the polynucleotide
encoding HemH; the polynucleotide encoding Heme-transporter; the
polynucleotide encoding the di-alpha chain; and the polynucleotide
encoding the beta chain are present in the host cell in one or more
plasmids.
[0095] In certain embodiments, the system for producing the
recombinant hemoglobin as described herein comprises: an
Escherichia coli or non-Escherichia coli host cell comprising: a
first plasmid comprising a polynucleotide encoding HemH, a di-alpha
chain, and two beta chains, wherein the di-alpha chain comprises a
polypeptide sequence having at least 98.93% sequence homology with
SEQ ID NO: 1, wherein the amino acids at position 1 and position
143 of SEQ ID NO: 1 must be methionine, the amino acids at position
29 and position 171 of SEQ ID NO: 1 must be phenylalanine, and the
amino acids at position 58 and position 200 of SEQ ID NO: 1 must be
glutamine; and the two beta chains comprises a polypeptide sequence
having at least 99.31% sequence homology with SEQ ID NO: 2, wherein
the amino acid at position 1 must be methionine; or a polypeptide
sequence having at least 97.94% sequence homology with SEQ ID NO:
3, wherein the amino acid at position 1 must be methionine, the
amino acid at position 82 must be aspartic acid, and the amino acid
at position 108 must be lysine; and a second plasmid comprising a
polynucleotide encoding Heme-transporter.
[0096] The vectors described herein can further comprise one or
more polynucleotides encoding a promoter, an operator, and a
selectable marker.
[0097] Any promoter can be used in the vectors described herein.
The selection of the appropriate promoter is well within the skill
of a person of ordinary skill in the art. Exemplary promoters
include, but are not limited to, lac, trp, tac, trc, ara, araB, T5,
T7, and T7lac.
[0098] Operators can include the lac operator, the .lamda.
operator, the trp operator, the gal operator, the ara operator, and
the Arg operator. If desired, the corresponding promoter may be
functionally associated with its operator.
[0099] A selectable marker can be a polynucleotide sequence that
allows selection for or against a molecule or a cell that contains
it, often under particular conditions. These markers can encode an
activity, such as, but not limited to, production of RNA, peptide,
or protein, or can provide a binding site for RNA, peptides,
proteins, inorganic and organic compounds or compositions and the
like. Any selectable marker in the art can be used in connection
with the expression systems and methods described herein. Exemplary
selectable markers include, but are not limited to, polynucleotide
sequences that encode products which provide resistance against
otherwise toxic compounds, such as antibiotics; polynucleotide
sequences that encode a product which is otherwise lacking in the
recipient cell, such as tRNA genes, auxotrophic markers);
polynucleotide sequences that encode a product which suppresses the
activity of a gene product; polynucleotide sequences that encode
products which can be readily identified (e.g., phenotypic markers,
such as .beta.-galactosidase, green fluorescent protein (GFP), and
cell surface proteins); and the like.
[0100] In certain embodiments, the selectable marker is a
polynucleotide sequence that confers resistance against an
antibacterial agent, such as ampicillin, kanamycin, erythromycin,
chloramphenicol, gentamycin, kasugamycin, rifampicin,
spectinomycin, D-Cycloserine, nalidixic acid, streptomycin,
tetracycline, and the like.
[0101] The present disclosure further relates to a method of
producing the recombinant hemoglobin as described herein,
comprising the steps of: (a) providing a host cell comprising a
polynucleotide encoding the di-alpha chain and a polynucleotide
encoding the two beta chains; and (b) inducing the host cell
containing the polynucleotide encoding the di-alpha chain and the
polynucleotide encoding the two beta chains to express recombinant
hemoglobin thereby producing the recombinant hemoglobin. In certain
embodiments, the polynucleotide encoding HemH; the polynucleotide
encoding Heme-transporter; the polynucleotide encoding the di-alpha
chain; and the polynucleotide encoding the two beta chains are
present in one or more vectors selected from the group consisting
of a plasmid, artificial chromosomes, viruses, and linear
polynucleotide fragments which are designed to insert into a cell's
genome. In certain embodiments, the polynucleotide encoding HemH;
the polynucleotide encoding Heme-transporter; the polynucleotide
encoding the di-alpha chain; and the polynucleotide encoding the
two beta chains are inserted into the host cell's genome using
CRISPR/CAS 9. In certain embodiments, the polynucleotide encoding
HemH; the polynucleotide encoding Heme-transporter; the
polynucleotide encoding the di-alpha chain; and the polynucleotide
encoding the two beta chains are present in the host cell in one or
more plasmids.
[0102] In certain embodiments, the method of producing the
recombinant hemoglobin described herein comprises: (a) providing a
host cell comprising one or more recombinant hemoglobin expression
plasmids encoding the di-alpha chain and the two beta chains; and
(b) inducing the host cell containing the one or more recombinant
hemoglobin expression plasmids encoding the di-alpha chain and the
two beta chains to express recombinant hemoglobin thereby producing
the recombinant hemoglobin.
[0103] In certain embodiments, the one or more recombinant
hemoglobin expression plasmids encoding the di-alpha chain and the
two beta chains comprise a polynucleotide sequence of SEQ ID NO: 4
and a polynucleotide sequence of SEQ ID NO: 5.
[0104] In certain embodiments, the one or more recombinant
hemoglobin expression plasmids encoding the di-alpha chain and the
two beta chains comprises a polynucleotide sequence of SEQ ID NO: 4
and a polynucleotide sequence of SEQ ID NO: 6.
[0105] In certain embodiments, the host cell is selected from the
group consisting of JM109 E. coli bacterial strain with lambda DE3,
BL21-AI E. coli bacterial strain without lambda DE3, and SHuffle E.
coli bacterial strain without lambda DE3.
[0106] In certain embodiments, the host cell further comprises a
plasmid encoding HemH.
[0107] In certain embodiments, the host cell further comprises one
or two plasmids encoding a Heme-transporter.
[0108] Also provided herein, is a host cell comprising at least one
vector described herein encoding at least one polypeptide selected
from a di-alpha chain as described herein and two beta chains as
described herein. In certain embodiments, the at least one vector
is a plasmid. In certain embodiments, the host cell comprises one
or more plasmids.
[0109] Recombinant hemoglobins can be formed by expressing one
di-alpha chain and two beta chains inside a host cell in the
presence of heme. Different host cells are tested and shown in
Table 3. In some embodiments, the host cell is JM109 E. coli
bacterial strain with lambda DE3, BL21-T7 or AI E. coli bacterial
strain without lambda DE3, or SHuffle E. coli bacterial strain
without lambda DE3 as they have the best yield, or
BL21-T7-CT(CRISPR/Cas9) and Jm109-T7-CT(CRISPR/Cas9) as they have
the best quality and easy manipulation. The expression of the
di-alpha chain and beta chains can be realized by co-transforming a
plasmid carrying the polynucleotide sequence that encodes one
di-alpha polypeptide chain, and a plasmid carrying the
polynucleotide sequence that encodes two beta polypeptide chains
together into the host cell, wherein the ratio of the plasmid for
di-alpha polypeptide chain and the plasmid for two beta polypeptide
chains is approximately 1:1. The expression of the di-alpha chain
and beta chains can also be realized by co-transforming a plasmid
carrying the polynucleotide sequence that encodes one di-alpha
polypeptide chain, and a plasmid carrying the polynucleotide
sequence that encodes one beta polypeptide chain together into the
host cell, wherein the ratio of the plasmid for di-alpha
polypeptide chain and the plasmid for one beta polypeptide chain is
approximately 1:2. The expression of the di-alpha chain and beta
chain can also be realized by transforming a plasmid carrying both
the polynucleotide sequence that encodes one di-alpha polypeptide
chain and the polynucleotide sequence that encodes two beta chains
into the host cells.
[0110] In certain embodiments, the plasmid for expressing the
recombinant hemoglobins described herein comprise a polynucleotide
encoding a polypeptide sequence having at least 98.93% sequence
homology with SEQ ID NO: 1, wherein the amino acids at position 1
and position 143 of SEQ ID NO: 1 must be methionine, the amino
acids at position 29 and position 171 of SEQ ID NO: 1 must be
phenylalanine, and the amino acids at position 58 and position 200
of SEQ ID NO: 1 must be glutamine; and two beta chains, each of
which comprises a polypeptide sequence having at least 97.94%
sequence homology with SEQ ID NO: 2, wherein position 1 of SEQ ID
NO: 2 must be methionine. In some embodiments, the plasmid for
expressing the recombinant hemoglobin comprises a polynucleotide
sequence of SEQ ID NO: 4 (di-alpha chain for TBM1 and TBM9) and a
polynucleotide sequence of SEQ ID NO: 5 (beta chain for TBM1). The
plasmid maps are shown in FIG. 2A. In certain embodiments, the host
cell is K12 strain (SHuffle.RTM. T7 Competent E. coli). In certain
embodiments, the plasmid for expressing the recombinant hemoglobin
comprises a polynucleotide sequence of SEQ ID NO: 4 (di-alpha chain
for TBM1 and TBM9) and a polynucleotide sequence of SEQ ID NO: 5
(beta chain for TBM1) and the host cell is K12 strain
(NEB#C3026).
[0111] In certain embodiments, the plasmid for expressing the
recombinant hemoglobins described herein comprise a polynucleotide
encoding a polypeptide sequence having at least 98.93% sequence
homology with SEQ ID NO: 1, wherein the amino acids at position 1
and position 143 of SEQ ID NO: 1 must be methionine, the amino
acids at position 29 and position 171 of SEQ ID NO: 1 must be
phenylalanine, and the amino acids at position 58 and position 200
of SEQ ID NO: 1 must be glutamine; and two beta chains, each of
which comprises a polypeptide sequence having at least 97.94%
sequence homology with SEQ ID NO: 3, wherein position 1 of SEQ ID
NO: 3 must be methionine, position 82 of SEQ ID NO: 3 must be
aspartic acid, and position 108 of SEQ ID NO: 3 must be lysine. In
certain embodiments, the plasmid for expressing the recombinant
hemoglobin comprises a polynucleotide sequence of SEQ ID NO: 4
(di-alpha chain for TBM1 and TBM9) and a polynucleotide sequence of
SEQ ID NO: 6 (beta chain for TBM9) (the plasmid maps are shown in
FIG. 2A) and the host cell is K12 strain (SHuffle.RTM. T7 Competent
E. coli).
[0112] In certain embodiments, the plasmids for expressing di-alpha
chain, beta chains and/or a combination thereof are co-transformed
with the plasmid for expressing ferrochelatase or any other enzyme
that catalyzes the production of heme. In certain embodiments, the
plasmid for expressing ferrochelatase or any other enzyme that
catalyzes the production of heme is a HemH plasmid, wherein the
HemH plasmid can contain any HemH nucleotide. An exemplary HemH
mutant polynucleotide is listed in FIG. 7. In certain embodiments,
the HemH plasmid has a polynucleotide sequence of SEQ ID NO: 7
(wildtype) or SEQ ID NO: 8 (mutant).
[0113] In certain embodiments, hemin is added into the solution
containing host cells that have been transformed with the plasmids
expressing di-alpha chain, beta chains and/or a combination
thereof.
[0114] In certain embodiments, a plasmid for expressing a heme
transporter is also co-transformed together with the plasmid for
expressing the recombinant hemoglobin and/or the plasmid for
expressing heme production enzymes. The heme transporter can be any
protein that can facilitate the uptake of heme, including but not
limited to a 69 kDa outer membrane protein encoded by the ChuA
gene, and an inner membrane protein named TonB, on which the uptake
of heme by ChuA is dependent. Any other heme and/or iron
transporters, such as the heme carrier protein 1 (HCP1), divalent
metal transporter 1 (DMT1), mucolipin-1 (also known as TRPML1),
HRG1, hemopexin, and the like are also within the contemplation of
the present disclosure.
[0115] In certain embodiments, the Heme-transporter plasmid
comprises a polynucleotide sequence having 98% sequence homology
with SEQ ID NO: 9. The DNA sequence of ChuA is shown in FIG. 8. In
certain embodiments, the Heme-transporter plasmid comprises a
polynucleotide sequence having 99% sequence homology with SEQ ID
NO: 9. In certain embodiments, the Heme-transporter plasmid
comprises a polynucleotide sequence of SEQ ID NO: 9.
[0116] In certain embodiments, the Heme-transporter plasmid
comprises a polynucleotide sequence having 98% sequence homology
with SEQ ID NO: 10. The DNA sequence encoding TonB is shown in FIG.
9. In certain embodiments, the Heme-transporter plasmid comprises a
polynucleotide sequence having 99% sequence homology with SEQ ID
NO: 10. In certain embodiment, the Heme-transporter plasmid
comprises a polynucleotide sequence of SEQ ID NO: 10.
[0117] In certain embodiments, only the Heme-transporter plasmid
bearing the ChuA is co-transformed with the plasmids for expressing
di-alpha chain, beta chains and/or a combination thereof In certain
embodiments, only the Heme-transporter plasmid bearing the ChuA is
co-transformed with the plasmids for expressing di-alpha chain,
beta chains and/or a combination thereof, and the plasmid for
expressing heme production enzymes. In certain embodiments, both
the plasmid bearing the ChuA gene and the plasmid for expressing
TonB are co-transformed with the plasmids for expressing di-alpha
chain, beta chains and/or a combination thereof. In certain
embodiments, both the plasmid bearing the ChuA gene and the plasmid
for expressing TonB are co-transformed with the plasmids for
expressing di-alpha chain, beta chains and/or a combination
thereof, and the plasmid for expressing heme production
enzymes.
[0118] In some embodiments, different protein expression systems
have been tested (shown in Table 4) and the protein expression
system for clone no. 12 and 19 show the best yield and quality of
recombinant hemoglobin. The pETDuet-1 vector has two T7 promoters.
The first T7 promoter is T1 and the second T7 promoter is T2. The
proteins can be expressed under the control of either T1 or T2 of
the pETDuet-1 vector. Similarly, the pRSFDuet-1 vector has two T7
promoters, and the first T7 promoter is R1 and the second T7
promoter is R2. The proteins can be expressed under the control of
either T1 or T2 of the pRSFDuet-1 vector.
TABLE-US-00004 TABLE 4 Different protein expression systems
Expressed proteins Clone Vectors and antibiotic Copy Heme No.
resistance Replicons Number Di - .alpha. + .beta. Ferrochelatase
transporter 1 pETDuet-1 (Ampicillin) ColE1 ~40 (T1) 2 (T2) 3 (T2)
(.beta. + Di-.alpha.) 4 (T2) (T1) 5 (T1) (T2) 6 (T2) M (T1) 7 (T1)
M (T2) 8 pRSFDuet-1 (Kanamycin) RSF1030 >100 (T2) 9 pETDuet-1
(Ampicillin) ColE1 ~40 (T1) (R2) 10 pRSFDuet-1 (Kanamycin) RSF1030
>100 (T2) (R2) 12 (T1) M (T2) (R2) 12 (T2) M (T1) (R2) 13
pETDuet-1 (Ampicillin) ColE1 ~40 (T2) M (T1) (A2) pACYCDuet-1 P15A
10-12 (Chloramphenicol) 14 pETDuet-1 (Ampicillin) ColE1 ~40 (T2) M
(T1) (C2) pCDFDuet-1 (Streptomycin) CloDF13 20-40 15 pRSFDuet-1
(Kanamycin) RSF1030 >100 (R2) M (R1) (A2) 16 pACYCDuet-1 P15A
10-12 (R1) M (R2) (A2) (Chloramphenicol) 17 pRSFDuet-1 (M) RSF1030
>100 (R1) M (R2) (A2) (Kanamycin) pACYCDuet-1 P15A 10-12
(Chloramphenicol) 18 pRSFDuet-1 (Kanamycin) RSF1030 >100 (R2) M
(R1) 19 pETDuet-1-Kana ColE1 ~40 (T2) M (T1) (A2) (Kanamycin)
pACYCDuet-1 P15A 10-12 (Chloramphenicol)
[0119] The components for the expression system contain (1) T7
promoter/lac operator and (2) sequence for protein expression
including start codon and stop codon. Each of Duet plasmid has two
T7 promoter/lac operator (T1 and T2). We inserted the different
genes under control of 1.sup.st or 2.sup.nd T7 promoter/lac
operator to test the protein expression level. The antibiotic
resistance and replicons of plasmid were shown in Table 4. Clone
no. 12 and 19 show the best yield and quality of recombinant
hemoglobin. For clone 12, it contained two plasmids: 1.sup.st
plasmid is pETDuet-1 (ampicillin) which expressed di-alpha and beta
chain under T1 and HemH under T2; 2nd plasmid is pRSFDuet-1
(kanamycin) which expressed ChuA and TonB under R2. For clone 19,
it contained two plasmids: 1.sup.st plasmid is pETDuet-1-Kana
(kanamycin) which expressed di-alpha and beta chain under T1 and
HemH under T2; 2.sup.nd plasmid is pACYCDuet-1 (chloramphenicol)
which expressed ChuA and TonB under A2.
[0120] The host cells containing one or more of the above-mentioned
plasmids can be incubated and fermented using any method known to
those of skill in the art. The recombinant hemoglobins produced by
the bacterial cells can then be purified using a simplified
procedure, which does not require a heat step, and does not require
the use of carbon monoxide (FIG. 6). The recombinant hemoglobins
can be purified using the purification procedure described herein
and can yield purified hemoglobin containing little or no HbCO, and
having low amounts of PPIX, e.g., less than 1%. The UPLC results
indicate that the heme: protein ratio is higher for TBM1 and TBM9,
and highest for TBM9 (Table 2). The High Performance Liquid
Chromatography (HPLC) results indicate the octamer: tetramer: dimer
distribution of TBN, TBM1 and TBM9, as shown in Table 5 below.
TABLE-US-00005 TABLE 5 HPLC results for different recombinant
hemoglobins Protein Octamer % Tetramer % Dimer % TBN 2.50 97.50
0.00 TBM1 0 100 0.00 TBM9 0 100 0.00
[0121] Purification of the recombinant hemoglobins described herein
can yield the protein product in high purity even without the step
of heat treatment, as shown in FIG. 4 and Tables 1 and 2. Such
unexpected technical effect solves one or more problems of the
existing methods for preparing recombinant hemoglobins. For
example, the stability of the recombinant hemoglobins described
herein during the purification will not be affected and thus CO
will not be required to maintain the stability of recombinant
hemoglobins during heat treatment. Accordingly, an additional step
to remove the undesired CO is not required. As such, the
purification process for the recombinant hemoglobins described
herein do not require heat treatment and CO removal, hence making
the process simpler, less costly, and more industrially
applicable.
[0122] The recombinant hemoglobins prepared using the methods
described herein were injected in a mouse model to measure the
liver oxygen level for 180 min (FIG. 3). Results showed that the
recombinant hemoglobin TBM1, wherein the di-alpha chain has SEQ ID
NO: 1 and the two beta chains have SEQ ID NO: 2, significantly
increased the liver oxygen pressure at 30 min after being
administered, as compared to the control group where buffer was
administered and the YQ (bovine crossed-linked hemoglobin, batch
no. ER007) was administered (FIG. 3B). The measurement of liver
oxygen pressure over 180 min shows that the liver oxygen pressure
reaches a plateau at 20-30 min after administering TBM9 and reaches
a plateau at about 70 min after administering TBM1, both of which
are higher than the plateaus reached by administering YQ, buffer,
or TBN (wherein the di-alpha chain has SEQ ID NO: 11, and the two
beta chains have SEQ ID NO: 12, as shown in FIG. 1). The p50 values
of TBN, TBM1, and TBM9 are summarized in Table 6.
TABLE-US-00006 TABLE 6 p50 values and oxidation resistance of TBN,
TBM1, and TBM9 Resistance to oxidation Recombinant hemoglobin p50
(mmHg) (shown in FIG. 10) TBN ~15 Normal (di-.alpha. = SEQ ID NO:
11; 2 .times. .beta. = SEQ ID NO: 12) TBM1 ~25 Improved (di-.alpha.
= SEQ ID NO: 1; 2 .times. .beta. = SEQ ID NO: 2) TBM9 ~40 Improved
(di-.alpha. = SEQ ID NO: 1; 2 .times. .beta. = SEQ ID NO: 3)
[0123] The recombinant hemoglobins described herein may be prepared
as a pharmaceutical composition comprising at least one
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers can comprise diluents, adjuvants, excipients, vehicles, or
a combination thereof with which the active compound is
administered. Such vehicles can be liquids, such as water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. For example, 0.4% saline and 0.3% glycine can be
used. In certain embodiments, such vehicle is sucrose or trehalose.
These solutions are sterile and generally free of particulate
matter. They may be sterilized by conventional, well-known
sterilization techniques (e.g., filtration). The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, stabilizing, thickening,
lubricating and coloring agents, etc. The concentration of the
protein in such pharmaceutical formulation can vary widely, e.g.,
from less than about 0.5%, usually at or at least about 1% to as
much as 15 or 20% by weight and will be selected primarily based on
required dose, fluid volumes, viscosities, etc., according to the
particular mode of administration selected. Suitable vehicles and
formulations comprise sucrose, trehalose, amino acid, phosphate
buffer.
[0124] The recombinant hemoglobins described herein can be used as
an agent for oxygenating in vivo or ex vivo tissues. Such agents
are useful in the treatment of any disease or condition in which
oxygen deficiency is the cause of the disease and/or condition, or
oxygen deficiency is related to the treatment efficacy of the
disease and/or condition. These diseases or conditions include, but
not limited to, cancer, stroke, hemorrhagic shock, AMS, PAD, PD
and, chronic obstructive pulmonary disease (COPD), emphysema,
bronchitis, pneumonia, pulmonary edema, anemia, asthma, heart
disease, diabetes, cystic fibrosis, epilepsy and seizures,
inflammatory bowel disease (IBD), and Raynaud's disease. The
required dosages of TBM1 and TBM9 can be significantly reduced as
demonstrated by animal studies described herein.
[0125] The present disclosure also provides expression clones that
can, e.g., improve the purity of recombinant hemoglobins described
herein. As shown in FIG. 4 and Table 7, the purified protein
expressed using the previous expression clone bearing nucleotide
sequences of HemH and TBN, TBM1, or TBM9 has a peak for PPIX in the
UPLC-PDA analysis, whereas such peak disappeared for the purified
protein expressed using the new improved expression clone bearing
nucleotide sequences of HemH mutant, Heme transporter system (ChuA
and TonB), and TBN, TBM1, or TBM9. This shows that the expression
clones described herein can significantly increase the percentage
of heme incorporation in the produced hemoglobin, which improves
the oxygen carrying function of the recombinant hemoglobins, and
significantly decreases the PPIX, which is a byproduct and major
impurity that can impair the oxygen carrying function of the
produced recombinant hemoglobins.
TABLE-US-00007 TABLE 7 UPLC-PDA analysis results of heme and PPIX
contents *PPIX % Standard (wt/wt) Deviation New clone (with HemH
mutant and heme 1.05 0.38 transporter system, ChuA + TonB): TBN Old
clone (With HemH wildtype, without heme 29.12 2.81 transporter
system, ChuA + TonB): TBN (*The PPIX % value is the average from
three batches)
[0126] To mimic in vivo conditions, the 2,3-DPG is added to the
recombinant hemoglobin solution. The p50 value of human recombinant
hemoglobin in the presence of 2,3-DPG increased as compared to the
human recombinant without 2,3-DPG (FIG. 5B).
EXAMPLE 1
Construction of TBN, TBM1, TBM9, HemH, ChuA, and TonB Expression
Plasmids
[0127] The plasmids were constructed in a conventional cloning
method and the vectors and restriction enzyme cute sites used for
each of the plasmids are indicated in the Table 8 below.
TABLE-US-00008 TABLE 8 Different plasmids information Plasmid
Vector Restriction Enzyme Name Insert Name Cut Site pACYCDuet- ChuA
+ TonB pACYCDuet- NdeI and PacI CT 1 pET-Hm- HemH(mutant); pETDuet-
XbaI and BamHI TBN-Kana TBN 1-kana for HemH mutant; NdeI and PacI
for TBN pET-Hm- HemH(mutant); pETDuet- XbaI and BamHI TBM1-Kana
TBM1 1-kana for HemH mutant; NdeI and PacI for TBM1 pET-Hm-
HemH(mutant); pETDuet- XbaI and BamHI TBM9-Kana TBM9 1-kana for
HemH mutant; NdeI and PacI for TBM9
EXAMPLE 2
Protein Expression
Small Scale Protein Expression: For Screening
[0128] The desired plasmids were transformed into the desired
strains. From a fresh culture plate, colonies were picked and
placed in a 15 ml tube containing 2 ml LB medium and appropriate
antibiotic (Kanamycin 50 .mu.g/ml; Chloramphenicol 34 .mu.g/ml) and
grown at 32.degree. C. overnight, 250 rpm. The resulting cultures
were diluted 1:100 into 10 ml TB medium with appropriate
antibiotics in 50 ml falcon. When OD.sub.600 reached 0.6, target
proteins were induced by 0.4 mM IPTG at 25.degree. C. After 20 h
induction, the bacteria were harvest by centrifugation at
4000.times. g for 15 min.
Fermentation
[0129] E. coli clones transformed with desired plasmids were
inoculated into 200 ml of seed culture medium (6% yeast extract and
1% NaCl) and cultivated at 32.degree. C. with shaking at 250 rpm
for overnight. The fermentation was performed in 10-L bioreactor
(Sartorius C plus). The flask of seed culture was inoculated into
6.5 L of medium (1% yeast extract, 1.6% tryptone, 15 mM
K.sub.2HPO.sub.4, 37 mM KH.sub.2PO.sub.4, 15 mM NaCl, 15 mM
(NH.sub.4).sub.2SO.sub.4, 2 mM L-proline, 2% glycerol) in a 10-L
bioreactor with a final OD.sub.600 at 0.05 and cultivated at
32.degree. C. and pH 7.0 with airflow of 6 L/min and initial stir
rate at 400 rpm, in which dissolved oxygen was maintained above
20%.
[0130] The fed-batch process of recombinant protein production in
10-L bioreactor was divided into two phases. The 1.sup.st phase was
aerobic batch cultivation at 32.degree. C. for 7 h. The 2.sup.nd
Phase was an induction process at 25.degree. C. for 18 h using
isopropyl-.beta.-d-thiogalactoside (IPTG) at a final concentration
of 0.4 mM with OD.sub.600 of bacteria at 4.0 to induce the
expression of recombinant protein. A fed-batch cultivation at
25.degree. C. for 18 h in which 80 g/L glycerol and 100 mg/L Hemin
was fed continuously to maintain the required specific growth rate
of bacteria with airflow of 8 L/min and stir rate at 600-800 rpm,
in which dissolved oxygen was maintained .about.4%. The bacteria at
final OD.sub.600 of 30-40 were harvested by centrifugation and
stored in -80.degree. C. for future use.
EXAMPLE 3
Protein Purification
Small Scale Protein Purification by Nickel-Beads: For Screening
[0131] Cell pellet from 10 ml TB medium was suspended in a
phosphate buffer (pH 7.4) and disrupted by 0.1 mm glass beads. The
soluble protein was harvested by centrifugation at 15000.times. g
for 15 min at 4.degree. C. The target protein bound to nickel
charged beads and eluted by 0.4 M Imidazole.
Protein Purification
Cell Lysis and Clarification of Crude Protein Sample
[0132] The cell pellet from fermentation was resuspended in a
solubilization buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.5) in 1 g
cell to 7 ml buffer ratio. The suspension was processed through a
high-pressure homogenizer at 800 bar (two cycles). The cell lysate
was clarified by hollow fiber system.
Chromatographic Purification
[0133] To remove impurities, the soluble fraction was passed
through a series of chromatographic columns to isolate the TBN,
TBM1, or TBM9 proteins.
[0134] First, the supernatant was loaded onto zinc affinity column.
After sufficient column wash with buffer (20 mM Tris, 30 mM NaCl,
pH 8.5), the target proteins were eluted by elution buffer (20 mM
Tris, 100 mM imidazole, pH 8.3).
[0135] Second, the recovered fraction was loaded onto DEAE
anion-exchange column. The target proteins were eluted by elution
buffer (20 mM NaPi, pH 6.0).
[0136] Finally, the purified TBN/TBM1/TBM9 samples were
concentrated to 50 mg/ml. The protein samples were lyophilized with
appropriate excipients in a glass vial.
EXAMPLE 4
Characterization of the Expressed Proteins
(a) UPLC
[0137] The protein concentrations of the samples were measured by
Bradford protein assay. Samples were diluted to protein
concentration of about 10 mg/ml. A 50 .mu.l of purified protein was
mixed with 400 .mu.l acidic acetone. After vigorous stirring, the
mixture was separated by centrifugation. Acetonitrile (ACN, 400
.mu.l, sample to organic solvent equals 1:8) was added into the
solution, followed by centrifugation at 14,000 rpm for 5 min.
Samples were then applied to UPLC analysis (Acquity H-class UPLC
system; Waters, Milford, Mass., USA).
[0138] PPIX was separated by Waters Acquity UPLC.RTM. BEH C18 1.7
.mu.m, 2.1.times.50 mm Column at 0.40 ml/min at 25.degree. C. for
15 min (Eluent: A: H.sub.2O; B: ACN [0.1% TFA]) Gradient: 30% B to
50% B in 6 min, 50% B to 80% B in 12 min, 80% B to 30% B in 13 min,
30% B in 15 min. (Detection wavelength at 400 nm). Commercially
obtained PPIX (Sigma-Aldrich, St. Louis, Mo., USA) was used as the
standard. The peak of PPIX was identified and the peak area in the
calibration standards and samples was recorded. The peak area of
calibration standards was plotted against the PPIX concentration of
working standards. Thus, the relative amount of PPIX was obtained
using the calibration curve and associated equation.
(b) High-Performance Liquid Chromatography (HPLC)
[0139] Samples were diluted to protein concentration about 1 mg/ml.
A 10 .mu.l of purified protein was applied to high-performance
liquid chromatography (HPLC) analysis (Waters Breeze2 HPLC system;
Waters, Milford, Mass., USA).
[0140] Proteins were separated by Yarra 3 .mu.m, SEC-2000,
7.8.times.300 mm Column at 0.50 ml/min at 25.degree. C. for 20 min
(Mobile phase: 0.75 M Magnesium Chloride, 0.2 M Tris, 116 .mu.M
EDTA). (Detection wavelength at 410 nm).
EXAMPLE 5
Measurement of Oxygen-Carrying Capacity of the Recombinant
Hemoglobins
(a) Oxyhemoglobin Dissociation Curve (ODC) and p50 Value
Measurement
[0141] p50 value was determined by HEMOX-analyzer. 1.5 mg of
purified protein were diluted in 3.5 ml of buffer (HEPES 100 mM,
KCl 100 mM, pH7.3) with or without 2,3-DPG (4 mM). The sample
buffer is drawn into a cuvette and the temperature of the mixture
is equilibrated and brought to 37.degree. C.; the sample is then
oxygenated to 100% with air. After adjustment of the pO.sub.2
value, the sample is deoxygenated with nitrogen. During the
deoxygenation process, the curve is recorded. The p50 value is
extrapolated on the x-axis as the point at which O.sub.2 saturation
is 50%.
(b) Liver Oxygen Level Measurement (180 Min) in a Rat Model
[0142] Liver tissue oxygenation (Liver pO.sub.2) was directly
monitored by the OxyLab.RTM. in vivo monitoring system (Oxford
Optronix, UK) during the ischemia and reperfusion procedures in
Buffalo rats. Briefly, a largearea-surface (LAS) oxygen sensor
(Oxford Optronix, UK) was placed between the right hepatic lobe and
triangle lobe of the rat livers. The branch of hepatic artery and
portal vein to right and triangle lobes were clamped. YQ (bovine
fumaryl cross-linked hemoglobin--positive control group) (0.2
g/kg), TBM1/TBM9 (0.2 g/kg), or ringer's acetate buffer (control
group) were administered intravenously. Liver oxygen tension was
continuously measured during hepatic ischemia reperfusion injury:
(1) baseline; (2) after infusion of YQ, TBM1/TBM9, or ringer's
acetate buffer.
EXAMPLE 6
Azide-Induced Oxidation Measurement
[0143] Recombinant hemoglobins were diluted to 1 mg/ml with 0.1 M
sodium phosphate plus 1 mM EDTA at pH 7.0 at room temperature.
Sample solution was oxygenated with compressed air by Hemox
Analyzer (TCS scientific) for 20 min at room temperature.
Azide-induced oxidation was started by adding 55.6 .mu.l of 2 M
sodium azide to 1 ml of oxygenated sample solution. The sample
solution was then transferred without delay into cuvettes, sealing
with parafilm to prevent evaporation. Visible spectra from 400 nm
to 800 nm were recorded by Multiskan GO with AuDROP plate (thermos
scientific) under room temperature in fast mode at 0, 1, 2, 3, 4,
5, 6, 24 and 48 h. Readings at 578 nm, 630 nm, and 700 nm were used
to calculate the concentration of oxy-Hb with the equation
below:
[oxy-Hb]=16.2*(A578-A700)-68*(A630-A700).
[0144] The initial rate of oxidation was calculated by an
exponential fitting of the first 6 h of data.
EXAMPLE 7
4T1 Breast Cancer Cell Proliferation Study
(a) 4T1 In Vitro Study
[0145] To determine the inhibition of cancer cell proliferation in
vitro, 4T1 breast cancer cells (2000 cells/well) were seeded onto
96-well plates and incubated for 24 h at 37.degree. C. After cell
stabilization, the culture medium was replaced with 100 ul of
culture medium containing TBM1 or TBM9, cell viability was
evaluated by MTT assay after 48 h. The results of the MTT assay on
the cell proliferation after TBM1 or TBM9 treatment were shown in
FIG. 11. TBM1 and TBM9 can significantly inhibit cancer cell
proliferation at the concentration from 0.1 to 10 uM.
(b) 4T1 In Vivo Study--Mouse
[0146] Six-week-old female BALB/c nude mice were subcutaneously
injected with 4T1 cancer cells (5000 cells per mice). Tumor volume
(mm.sup.3) was calculated according to the following standard
formula: (the longest diameter).times.0.5.times.(the shortest
diameter). After the tumor volume reached 100 mm.sup.3, TBM1
protein (100 mg/kg, n=8) was injected by tail vein once per week
for two weeks. The control group (n=8) was injected PBS. The tumor
volume was monitored twice per week. As shown in FIG. 12A, tumors
grew rapidly in mice treated with PBS (control group) in about 14
days after the treatment. Tumor growth was slowed down
significantly in mice in TBM1 group compared with control groups
(p<0.05). FIG. 12B showed tumor volume after 14 days treatments
of TBM1. For control group, tumor reached a volume of 800 mm.sup.3
which was substantially higher than that of the TBM1 treatment
group (377 mm.sup.3).
EXAMPLE 8
Peripheral Arterial Disease (PAD) Model
[0147] A murine model was used to evaluate the potential
therapeutic effect of TBM1 on critical limb ischemia. Briefly, mice
were anesthetized by intraperitoneal injection of xylazine (20
mg/kg) and ketamine (100 mg/kg). The left femoral artery was
excised and ligated from its proximal origin as a branch of the
external iliac artery to the distal point where it bifurcates into
the saphenous and popliteal arteries. 24 hours after successful
femoral artery ligation, mice were randomly allocated to receive a
single intravenous administration of normal saline (n=2) and TBM1
(800 mg/kg, n=2) via the tail veins.
[0148] Tissue perfusion of the hind limbs were assessed serially at
baseline, immediate after femoral artery ligation, and on Day 7
after administration of the allocated treatments by using laser
Doppler imaging system (Moor instruments, Devon, UK). Mice were
anesthetized during the measurement procedure. The digital
color-coded images were captured and analyzed to quantify blood
flow in the region from the knee joint to the toes.
[0149] As shown in FIG. 13, In the time point of "immediate after
right femoral artery ligation", our results showed the mean
relative blood perfusion reduced substantially from 88.8% to only
5.2% of that the non-ischemic control limbs, confirming the
successful induction of acute hind-limb ischemia. Interestingly,
there was a large improvement in tissue blood perfusion in the
ischemic limbs of mice receiving TBM1 treatment on Day 7.
Specifically, the mean relative tissue blood perfusion was 11.7% of
that of the non-ischemic control limbs in the normal saline group
at Day 7, which was substantially lower than that of the
counterparts receiving TBM1 at 800 mg/kg (30.1%).
[0150] In conclusion, single intravenous TBM1 injection at 800
mg/kg results in an improvement of tissue perfusion in a murine
model of critical limb ischemia.
EXAMPLE 9
Multiple Dose and Single Dose of TBM1 in Mice & Rat
(a) Multiple Dose of TBM1 in Mice
[0151] Mice (both Balb/C mice and ICR mice) were randomly divided
into 3 groups (n=6) and received 4 consecutive weekly injections
(TBM1 100, 200 mg/kg or PBS as a control) through the tail vein. At
the endpoint, the mice were anesthetized and the blood samples were
collected and analyzed for hematology analysis. The major organs
(liver, spleen, kidney, lung) were harvested, fixed in formalin and
processed for histological evaluation (H&E staining).
[0152] As shown in the FIG. 14, no significant differences in
weights were seen between the TBM1 treatment groups and control
group (both for Balb/C mice and ICR mice). Other usual signs of
drug toxicity in mice, such as ruffled fur, anorexia, cachexia,
skin tenting (due to dehydration), skin ulcerations, or toxic
deaths were not seen at the doses used.
(b) Single Dose of TBM1 in Rat (for Checking Cardiomyopathy)
[0153] SD rats (n=6) were received single dose (TBM1 400 and 800
mg/kg, by intravenous infusion). Briefly, jugular catheter was
surgically implanted into the right jugular vein of the rats at
least 4 days before the dosing day. On the dosing day, animals were
weighted and assigned into 6 per groups with half numbers of male
and female. TBM1 was drawn into syringe and connected to an
extension tube with a 0.22 um filter for injection. The infusion
was given at a constant rate by motorized syringe pump. After 72 h,
the rats were sacrificed and the hearts were harvested, fixed in
formalin and processed for histological evaluation (H&E
staining). There was no evidence of drug induced cardiomyopathy in
total six rats.
[0154] Although the invention has been described in terms of
certain embodiments, other embodiments apparent to those of
ordinary skill in the art are also within the scope of this
invention. Accordingly, the scope of the invention is intended to
be defined only by the claims which follow.
INDUSTRIAL APPLICABILITY
[0155] The present disclosure provides recombinant hemoglobins
useful for treating oxygen deficiency related diseases or
conditions, such as cancer, stroke, hemorrhagic shock, acute
mountain sickness (AMS), peripheral arterial disease (PAD) and
Parkinson's disease (PD). The recombinant hemoglobins described
herein can have increased oxygen carrying capacity. The therapeutic
use of the pharmaceutical compositions comprising the recombinant
hemoglobins described herein can require a reduced clinical dosage,
which increases the safety of such therapeutic use in a subject in
need thereof. Also provided are simplified methods and/or processes
to prepare, obtain, ferment, and/or purify the recombinant
hemoglobins with high yield and high purity. Such methods can be
used at an industrial scale, which can be more cost effective and
less impurity-prone as compared to existing conventional methods.
Sequence CWU 1
1
151283PRTArtificial Sequencedi-alpha chain, synthesized in lab 1Met
Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys1 5 10
15Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Phe Glu Arg Met
20 25 30Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp
Leu 35 40 45Ser His Gly Ser Ala Gln Val Lys Gly Gln Gly Lys Lys Val
Ala Asp 50 55 60Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro
Asn Ala Leu65 70 75 80Ser Ala Leu Ser Asp Leu His Ala His Lys Leu
Arg Val Asp Pro Val 85 90 95Asn Phe Lys Leu Leu Ser His Cys Leu Leu
Val Thr Leu Ala Ala His 100 105 110Leu Pro Ala Glu Phe Thr Pro Ala
Val His Ala Ser Leu Asp Lys Phe 115 120 125Leu Ala Ser Val Ser Thr
Val Leu Thr Ser Lys Tyr Arg Gly Met Leu 130 135 140Ser Pro Ala Asp
Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly145 150 155 160Ala
His Ala Gly Glu Tyr Gly Ala Glu Ala Phe Glu Arg Met Phe Leu 165 170
175Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu Ser His
180 185 190Gly Ser Ala Gln Val Lys Gly Gln Gly Lys Lys Val Ala Asp
Ala Leu 195 200 205Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn
Ala Leu Ser Ala 210 215 220Leu Ser Asp Leu His Ala His Lys Leu Arg
Val Asp Pro Val Asn Phe225 230 235 240Lys Leu Leu Ser His Cys Leu
Leu Val Thr Leu Ala Ala His Leu Pro 245 250 255Ala Glu Phe Thr Pro
Ala Val His Ala Ser Leu Asp Lys Phe Leu Ala 260 265 270Ser Val Ser
Thr Val Leu Thr Ser Lys Tyr Arg 275 2802146PRTArtificial
Sequencebeta chain, synthesized in lab 2Met His Leu Thr Pro Glu Glu
Lys Ser Ala Val Thr Ala Leu Trp Gly1 5 10 15Lys Val Asn Val Asp Glu
Val Gly Gly Glu Ala Leu Gly Arg Leu Leu 20 25 30Val Val Tyr Pro Trp
Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu 35 40 45Ser Thr Pro Asp
Ala Val Met Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60Lys Lys Val
Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn65 70 75 80Leu
Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu 85 90
95His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Cys
100 105 110Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val
Gln Ala 115 120 125Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala
Leu Ala His Lys 130 135 140Tyr His1453146PRTArtificial Sequencebeta
chain, synthesized in lab 3Met His Leu Thr Pro Glu Glu Lys Ser Ala
Val Thr Ala Leu Trp Gly1 5 10 15Lys Val Asn Val Asp Glu Val Gly Gly
Glu Ala Leu Gly Arg Leu Leu 20 25 30Val Val Tyr Pro Trp Thr Gln Arg
Phe Phe Glu Ser Phe Gly Asp Leu 35 40 45Ser Thr Pro Asp Ala Val Met
Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60Lys Lys Val Leu Gly Ala
Phe Ser Asp Gly Leu Ala His Leu Asp Asn65 70 75 80Leu Asp Gly Thr
Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu 85 90 95His Val Asp
Pro Glu Asn Phe Arg Leu Leu Gly Lys Val Leu Val Cys 100 105 110Val
Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln Ala 115 120
125Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys
130 135 140Tyr His1454852DNAArtificial Sequencedi-alpha chain,
synthesized in lab 4atgctgtctc cggcagataa aacgaatgtt aaagcagcct
ggggtaaagt cggcgctcac 60gcgggcgaat acggtgcaga agcctttgaa cgtatgtttc
tgtccttccc gaccacgaaa 120acctattttc cgcatttcga tctgagtcac
ggctccgcgc aggtgaaagg tcagggcaaa 180aaagttgcag acgctctgac
gaacgcggtg gcccacgttg atgacatgcc gaatgcactg 240tcagctctga
gcgatctgca cgcgcacaaa ctgcgtgttg atccggtgaa ctttaaactg
300ctgagccatt gcctgctggt caccctggcg gcacacctgc cggcagaatt
tacgccggcg 360gttcatgcca gcctggataa attcctggcg agcgtgagca
ccgtcctgac gtctaaatac 420cgcggcatgc tgtctccggc agataaaacg
aatgttaaag cagcctgggg taaagtcggc 480gctcacgcgg gcgaatacgg
tgcagaagcc tttgaacgta tgtttctgtc cttcccgacc 540acgaaaacct
attttccgca tttcgatctg agtcacggct ccgcgcaggt gaaaggtcag
600ggcaaaaaag ttgcagacgc tctgacgaac gcggtggccc acgttgatga
catgccgaat 660gcactgtcag ctctgagcga tctgcacgcg cacaaactgc
gtgttgatcc ggtgaacttt 720aaactgctga gccattgcct gctggtcacc
ctggcggcac acctgccggc agaatttacg 780ccggcggttc atgccagcct
ggataaattc ctggcgagcg tgagcaccgt cctgacgtct 840aaataccgct aa
8525441DNAArtificial Sequencebeta chain, synthesized in lab
5atgcatctga ccccggaaga aaaatcggct gtgacggctc tgtggggcaa agttaatgtg
60gacgaagtcg gcggcgaagc actgggccgt ctgctggttg tgtatccgtg gacccagcgc
120tttttcgaat cttttggtga tctgtctacg ccggacgctg ttatgggcaa
cccgaaagtc 180aaagcgcatg gcaaaaaagt cctgggtgcc tttagtgatg
gcctggcaca cctggacaat 240ctgaaaggca ccttcgcgac gctgagcgaa
ctgcattgcg ataaactgca cgtggacccg 300gaaaacttcc gtctgctggg
taatgttctg gtctgtgtgc tggcccatca ctttggcaaa 360gaatttaccc
cgccggtgca ggcggcctat caaaaagtcg tggcaggcgt tgcaaacgct
420ctggcgcata aataccacta a 4416441DNAArtificial Sequencebeta chain,
synthesized in lab 6atgcatctga ccccggaaga aaaatcggct gtgacggctc
tgtggggcaa agttaatgtg 60gacgaagtcg gcggcgaagc actgggccgt ctgctggttg
tgtatccgtg gacccagcgc 120tttttcgaat cttttggtga tctgtctacg
ccggacgctg ttatgggcaa cccgaaagtc 180aaagcgcatg gcaaaaaagt
cctgggtgcc tttagtgatg gcctggcaca cctggacaat 240ctggacggca
ccttcgcgac gctgagcgaa ctgcattgcg ataaactgca cgtggacccg
300gaaaacttcc gtctgctggg taaagttctg gtctgtgtgc tggcccatca
ctttggcaaa 360gaatttaccc cgccggtgca ggcggcctat caaaaagtcg
tggcaggcgt tgcaaacgct 420ctggcgcata aataccacta a
4417933DNAArtificial SequenceHemH wildtype, synthesized in lab
7atgtcgcgta agaagatggg attgctggtg atggcttacg ggacgcctta caaagaagag
60gacattgagc gttactatac gcacatccgt cgtgggcgta agcctgagcc tgagatgctt
120caggatctga aagatcgcta cgaggctatc gggggtattt ctcctttggc
acagattacc 180gaacaacaag cccataatct tgaacaacat ctgaatgaaa
ttcaagacga aattaccttc 240aaggcctaca ttggcttgaa acatattgag
ccttttatcg aggatgccgt ggctgaaatg 300cataaggatg gaatcaccga
agccgtttct atcgtgctgg ccccacattt ttccacgttc 360tcagtacaaa
gctacaataa acgcgcaaaa gaagaagccg agaagcttgg gggcttgaca
420atcacctctg tcgaatcatg gtatgatgaa ccgaaattcg ttacctattg
ggttgaccgt 480gtaaaggaga cctatgccag tatgcccgag gatgaacgcg
aaaacgccat gttaatcgtt 540agtgctcact cgctaccgga gaaaatcaaa
gaatttggtg acccgtaccc tgaccagctt 600cacgagtcgg caaaactgat
tgccgaagga gcgggtgttt cggaatacgc tgttggttgg 660caaagtgagg
gaaatacgcc agacccttgg ctgggtccgg atgtccaaga cttaacccgt
720gatctgttcg agcagaaagg ttatcaggcg ttcgtatatg tgcccgtggg
cttcgtggct 780gatcatctgg aggtcttgta tgataatgac tatgagtgca
aggtggtcac agacgatatc 840ggggcatcgt attatcgccc ggagatgcca
aatgctaaac cggaatttat tgacgccttg 900gctacggttg tacttaaaaa
acttggccgc taa 9338933DNAArtificial SequenceHemH mutant,
synthesized in lab 8atgtcgcgta agaagatggg attgctggtg atggcttacg
ggacgcctta caaagaagag 60gacattgagc gttactatac gcacatccgt cgtgggcgta
agcctgagcc tgagatgctt 120caggatctga aagatcgcta cgaggctatc
gggggtattt ctcctttggc acagattacc 180aaacaacaag cccataatct
tgaacaacat ctgaatgaaa ttcaagacga aattaccttc 240aaggcctaca
ttggcttgaa acatattgag ccttttatcg aggatgccgt ggctgaaatg
300cataaggatg gaatcaccga agccgtttct atcgtgctgg ccccacattt
ttccacgttc 360tcagtacaaa gctacaataa acgcgcaaaa gaagaagccg
agaagcttgg gggcttgaca 420atcacctctg tcgaatcatg gtatgatgaa
ccgaaattcg ttacctattg ggttgaccgt 480gtaaaggaga cctatgccag
tatgcccgag gatgaacgcg aaaacgccat gttaatcgtt 540agtgctcact
cgcaaccgga gaaaatcaaa gaattcggtg acccgtaccc tgaccagctt
600cacgagtcgg caaaactgat tgccgaagga gcggatgttt cggaatacgc
tgttggttgg 660caaagtgagg gaaatacgcc agacccttgg ctgggtccgg
atgtccaaga cttaacccgt 720gatctgttcg agcagaaagg ttatcaggcg
ttcgtatatg tgcccgtggg cttcgtggct 780gatcatctgg aggtcttgta
tgataatgac tatgagtgca aggtggtcac agacgatatc 840ggggcatcgt
attatcgccc ggagatgcca aatgctaaac cggaattcat tgacgccttg
900gctacggttg tacttaaaaa acttggccgc taa 93391983DNAArtificial
SequenceChuA, Synthesized in lab 9atgtcacgtc cgcaatttac ctcgttgcgt
ttgagtttat tggccttagc tgtttctgcc 60accttgccaa cgtttgcttt tgctactgaa
accatgaccg ttacggcaac ggggaatgcc 120cgtagttcct tcgaagcgcc
tatgatggtc agcgtcatcg acacttccgc tcctgaaaat 180caaacggcta
cttcagccac cgatctgctg cgtcatgttc ctggaattac tctggatggt
240accggacgaa ccaacggtca ggatgtaaat atgcgtggct atgatcatcg
cggcgtgctg 300gttcttgtcg atggtgttcg tcagggaacg gataccggac
acctgaatgg cacttttctc 360gatccggcgc tgatcaagcg tgttgagatt
gttcgtggac cttcagcatt actgtatggc 420agtggcgcgc tgggtggagt
gatctcctac gatacggtcg atgcaaaaga tttattgcag 480gaaggacaaa
gcagtggttt tcgtgtcttt ggtactggcg gcacggggga ccatagcctg
540ggattaggcg cgagcgcgtt tgggcgaact gaaaatctgg atggtattgt
ggcctggtcc 600agtcgcgatc ggggtgattt acgccagagc aatggtgaaa
ccgcgccgaa tgacgagtcc 660attaataaca tgctggcgaa agggacctgg
caaattgatt cagcccagtc tctgagcggt 720ttagtgcgtt actacaacaa
cgacgcgcgt gaaccaaaaa atccgcagac cgttggggct 780tctgaaagca
gcaacccgat ggttgatcgt tcaacaattc aacgcgatgc gcagctttct
840tataaactcg ccccgcaggg caacgactgg ttaaatgcag atgcaaaaat
ttattggtcg 900gaagtccgta ttaatgcgca aaacacgggg agttccggcg
agtatcgtga acagataaca 960aaaggagcca ggctggagaa ccgttccact
ctctttgccg acagtttcgc ttctcactta 1020ctgacgtatg gcggtgagta
ttatcgtcag gaacaacatc cgggcggcgc gacgacgggc 1080ttcccgcaag
caaaaatcga ttttagctcc ggctggctac aggatgagat caccttacgc
1140gatctgccga ttaccctgct tggcggaacc cgctatgaca gttatcgcgg
tagcagtgac 1200ggttacaaag atgttgatgc cgacaaatgg tcatctcgtg
cggggatgac tatcaatccg 1260actaactggc tgatgttatt tggctcttat
gcccaggcat tccgcgcccc gacgatgggc 1320gaaatgtata acgattctaa
gcacttctcg attggtcgct tctataccaa ctattgggtg 1380ccaaacccga
acttacgtcc ggaaactaac gaaactcagg agtacggttt tgggctgcgt
1440tttgatgacc tgatgttgtc caatgatgct ctggaattta aagccagcta
ctttgatacc 1500aaagcgaagg attacatctc cacgaccgtc gatttcgcgg
cggcgacgac tatgtcgtat 1560aacgtcccga acgccaaaat ctggggctgg
gatgtgatga cgaaatatac cactgatctg 1620tttagccttg atgtggccta
taaccgtacc cgcggcaaag acaccgatac cggcgaatac 1680atctccagca
ttaacccgga tactgttacc agcactctga atattccgat cgctcacagt
1740ggcttctctg ttgggtgggt tggtacgttt gccgatcgct caacacatat
cagcagcagt 1800tacagcaaac aaccaggcta tggcgtgaat gatttctacg
tcagttatca aggacaacag 1860gcgctcaaag gtatgaccac tactttggtg
ttgggtaacg ctttcgacaa agagtactgg 1920tcgccgcaag gcatcccaca
ggatggtcgt aacggaaaaa ttttcgtgag ttatcaatgg 1980taa
198310720DNAArtificial SequenceTonB, Synthesized in lab
10atgacccttg atttacctcg ccgcttcccc tggccgacgt tactttcggt ctgcattcat
60ggtgctgttg tggcgggtct gctctatacc tcggtacatc aggttattga actacctgcg
120cctgcgcagc cgatttctgt cacgatggtt gcgcctgctg atctcgaacc
gccacaagcc 180gttcagccgc caccggagcc ggtggtagag ccagaaccgg
aacctgagcc gatccccgaa 240ccgccaaaag aagcaccggt ggtcattgaa
aagccgaagc cgaaacctaa gccaaaaccg 300aagccggtga aaaaggtaca
ggagcagcaa aaacgcgatg tcaaacccgt agagtcgcgt 360ccggcatcac
cgtttgaaaa tacggcaccg gcacgcccga catcaagtac agcaacggct
420gcaaccagca agccggttac cagtgtggct tcaggaccac gcgcattaag
ccgtaatcag 480ccgcagtatc cggcacgagc acaggcattg cgcattgaag
ggcaggttaa agttaaattt 540gacgtcacgc cggatggtcg cgtggataac
gtacaaatcc tctcagccaa gcctgcgaac 600atgtttgagc gtgaggtgaa
aaatgcgatg cgcagatggc gttatgagcc gggtaagcca 660ggcagtggga
ttgtggtgaa tatcctgttt aaaattaacg gcaccaccga aattcagtaa
72011283PRTArtificial SequenceDi-alpha chain, Synthesized in lab
11Met Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys1
5 10 15Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg
Met 20 25 30Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe
Asp Leu 35 40 45Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys
Val Ala Asp 50 55 60Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met
Pro Asn Ala Leu65 70 75 80Ser Ala Leu Ser Asp Leu His Ala His Lys
Leu Arg Val Asp Pro Val 85 90 95Asn Phe Lys Leu Leu Ser His Cys Leu
Leu Val Thr Leu Ala Ala His 100 105 110Leu Pro Ala Glu Phe Thr Pro
Ala Val His Ala Ser Leu Asp Lys Phe 115 120 125Leu Ala Ser Val Ser
Thr Val Leu Thr Ser Lys Tyr Arg Gly Met Leu 130 135 140Ser Pro Ala
Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly145 150 155
160Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met Phe Leu
165 170 175Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu
Ser His 180 185 190Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val
Ala Asp Ala Leu 195 200 205Thr Asn Ala Val Ala His Val Asp Asp Met
Pro Asn Ala Leu Ser Ala 210 215 220Leu Ser Asp Leu His Ala His Lys
Leu Arg Val Asp Pro Val Asn Phe225 230 235 240Lys Leu Leu Ser His
Cys Leu Leu Val Thr Leu Ala Ala His Leu Pro 245 250 255Ala Glu Phe
Thr Pro Ala Val His Ala Ser Leu Asp Lys Phe Leu Ala 260 265 270Ser
Val Ser Thr Val Leu Thr Ser Lys Tyr Arg 275 28012146PRTArtificial
Sequencebeta chain, Synthesized in lab 12Met His Leu Thr Pro Glu
Glu Lys Ser Ala Val Thr Ala Leu Trp Gly1 5 10 15Lys Val Asn Val Asp
Glu Val Gly Gly Glu Ala Leu Gly Arg Leu Leu 20 25 30Val Val Tyr Pro
Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu 35 40 45Ser Thr Pro
Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60Lys Lys
Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn65 70 75
80Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu
85 90 95His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val
Cys 100 105 110Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro
Val Gln Ala 115 120 125Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn
Ala Leu Ala His Lys 130 135 140Tyr His14513310PRTArtificial
SequenceHemH mutant, synthesized in the lab 13Met Ser Arg Lys Lys
Met Gly Leu Leu Val Met Ala Tyr Gly Thr Pro1 5 10 15Tyr Lys Glu Glu
Asp Ile Glu Arg Tyr Tyr Thr His Ile Arg Arg Gly 20 25 30Arg Lys Pro
Glu Pro Glu Met Leu Gln Asp Leu Lys Asp Arg Tyr Glu 35 40 45Ala Ile
Gly Gly Ile Ser Pro Leu Ala Gln Ile Thr Lys Gln Gln Ala 50 55 60His
Asn Leu Glu Gln His Leu Asn Glu Ile Gln Asp Glu Ile Thr Phe65 70 75
80Lys Ala Tyr Ile Gly Leu Lys His Ile Glu Pro Phe Ile Glu Asp Ala
85 90 95Val Ala Glu Met His Lys Asp Gly Ile Thr Glu Ala Val Ser Ile
Val 100 105 110Leu Ala Pro His Phe Ser Thr Phe Ser Val Gln Ser Tyr
Asn Lys Arg 115 120 125Ala Lys Glu Glu Ala Glu Lys Leu Gly Gly Leu
Thr Ile Thr Ser Val 130 135 140Glu Ser Trp Tyr Asp Glu Pro Lys Phe
Val Thr Tyr Trp Val Asp Arg145 150 155 160Val Lys Glu Thr Tyr Ala
Ser Met Pro Glu Asp Glu Arg Glu Asn Ala 165 170 175Met Leu Ile Val
Ser Ala His Ser Gln Pro Glu Lys Ile Lys Glu Phe 180 185 190Gly Asp
Pro Tyr Pro Asp Gln Leu His Glu Ser Ala Lys Leu Ile Ala 195 200
205Glu Gly Ala Asp Val Ser Glu Tyr Ala Val Gly Trp Gln Ser Glu Gly
210 215 220Asn Thr Pro Asp Pro Trp Leu Gly Pro Asp Val Gln Asp Leu
Thr Arg225 230 235 240Asp Leu Phe Glu Gln Lys Gly Tyr Gln Ala Phe
Val Tyr Val Pro Val 245 250 255Gly Phe Val Ala Asp His Leu Glu Val
Leu Tyr Asp Asn Asp Tyr Glu 260 265 270Cys Lys Val Val Thr Asp Asp
Ile Gly Ala Ser Tyr Tyr Arg Pro Glu 275 280
285Met Pro Asn Ala Lys Pro Glu Phe Ile Asp Ala Leu Ala Thr Val Val
290 295 300Leu Lys Lys Leu Gly Arg305 31014660PRTArtificial
SequenceChuA, Synthesized in lab 14Met Ser Arg Pro Gln Phe Thr Ser
Leu Arg Leu Ser Leu Leu Ala Leu1 5 10 15Ala Val Ser Ala Thr Leu Pro
Thr Phe Ala Phe Ala Thr Glu Thr Met 20 25 30Thr Val Thr Ala Thr Gly
Asn Ala Arg Ser Ser Phe Glu Ala Pro Met 35 40 45Met Val Ser Val Ile
Asp Thr Ser Ala Pro Glu Asn Gln Thr Ala Thr 50 55 60Ser Ala Thr Asp
Leu Leu Arg His Val Pro Gly Ile Thr Leu Asp Gly65 70 75 80Thr Gly
Arg Thr Asn Gly Gln Asp Val Asn Met Arg Gly Tyr Asp His 85 90 95Arg
Gly Val Leu Val Leu Val Asp Gly Val Arg Gln Gly Thr Asp Thr 100 105
110Gly His Leu Asn Gly Thr Phe Leu Asp Pro Ala Leu Ile Lys Arg Val
115 120 125Glu Ile Val Arg Gly Pro Ser Ala Leu Leu Tyr Gly Ser Gly
Ala Leu 130 135 140Gly Gly Val Ile Ser Tyr Asp Thr Val Asp Ala Lys
Asp Leu Leu Gln145 150 155 160Glu Gly Gln Ser Ser Gly Phe Arg Val
Phe Gly Thr Gly Gly Thr Gly 165 170 175Asp His Ser Leu Gly Leu Gly
Ala Ser Ala Phe Gly Arg Thr Glu Asn 180 185 190Leu Asp Gly Ile Val
Ala Trp Ser Ser Arg Asp Arg Gly Asp Leu Arg 195 200 205Gln Ser Asn
Gly Glu Thr Ala Pro Asn Asp Glu Ser Ile Asn Asn Met 210 215 220Leu
Ala Lys Gly Thr Trp Gln Ile Asp Ser Ala Gln Ser Leu Ser Gly225 230
235 240Leu Val Arg Tyr Tyr Asn Asn Asp Ala Arg Glu Pro Lys Asn Pro
Gln 245 250 255Thr Val Gly Ala Ser Glu Ser Ser Asn Pro Met Val Asp
Arg Ser Thr 260 265 270Ile Gln Arg Asp Ala Gln Leu Ser Tyr Lys Leu
Ala Pro Gln Gly Asn 275 280 285Asp Trp Leu Asn Ala Asp Ala Lys Ile
Tyr Trp Ser Glu Val Arg Ile 290 295 300Asn Ala Gln Asn Thr Gly Ser
Ser Gly Glu Tyr Arg Glu Gln Ile Thr305 310 315 320Lys Gly Ala Arg
Leu Glu Asn Arg Ser Thr Leu Phe Ala Asp Ser Phe 325 330 335Ala Ser
His Leu Leu Thr Tyr Gly Gly Glu Tyr Tyr Arg Gln Glu Gln 340 345
350His Pro Gly Gly Ala Thr Thr Gly Phe Pro Gln Ala Lys Ile Asp Phe
355 360 365Ser Ser Gly Trp Leu Gln Asp Glu Ile Thr Leu Arg Asp Leu
Pro Ile 370 375 380Thr Leu Leu Gly Gly Thr Arg Tyr Asp Ser Tyr Arg
Gly Ser Ser Asp385 390 395 400Gly Tyr Lys Asp Val Asp Ala Asp Lys
Trp Ser Ser Arg Ala Gly Met 405 410 415Thr Ile Asn Pro Thr Asn Trp
Leu Met Leu Phe Gly Ser Tyr Ala Gln 420 425 430Ala Phe Arg Ala Pro
Thr Met Gly Glu Met Tyr Asn Asp Ser Lys His 435 440 445Phe Ser Ile
Gly Arg Phe Tyr Thr Asn Tyr Trp Val Pro Asn Pro Asn 450 455 460Leu
Arg Pro Glu Thr Asn Glu Thr Gln Glu Tyr Gly Phe Gly Leu Arg465 470
475 480Phe Asp Asp Leu Met Leu Ser Asn Asp Ala Leu Glu Phe Lys Ala
Ser 485 490 495Tyr Phe Asp Thr Lys Ala Lys Asp Tyr Ile Ser Thr Thr
Val Asp Phe 500 505 510Ala Ala Ala Thr Thr Met Ser Tyr Asn Val Pro
Asn Ala Lys Ile Trp 515 520 525Gly Trp Asp Val Met Thr Lys Tyr Thr
Thr Asp Leu Phe Ser Leu Asp 530 535 540Val Ala Tyr Asn Arg Thr Arg
Gly Lys Asp Thr Asp Thr Gly Glu Tyr545 550 555 560Ile Ser Ser Ile
Asn Pro Asp Thr Val Thr Ser Thr Leu Asn Ile Pro 565 570 575Ile Ala
His Ser Gly Phe Ser Val Gly Trp Val Gly Thr Phe Ala Asp 580 585
590Arg Ser Thr His Ile Ser Ser Ser Tyr Ser Lys Gln Pro Gly Tyr Gly
595 600 605Val Asn Asp Phe Tyr Val Ser Tyr Gln Gly Gln Gln Ala Leu
Lys Gly 610 615 620Met Thr Thr Thr Leu Val Leu Gly Asn Ala Phe Asp
Lys Glu Tyr Trp625 630 635 640Ser Pro Gln Gly Ile Pro Gln Asp Gly
Arg Asn Gly Lys Ile Phe Val 645 650 655Ser Tyr Gln Trp
66015239PRTArtificial SequenceTonB, Synthesized in lab 15Met Thr
Leu Asp Leu Pro Arg Arg Phe Pro Trp Pro Thr Leu Leu Ser1 5 10 15Val
Cys Ile His Gly Ala Val Val Ala Gly Leu Leu Tyr Thr Ser Val 20 25
30His Gln Val Ile Glu Leu Pro Ala Pro Ala Gln Pro Ile Ser Val Thr
35 40 45Met Val Ala Pro Ala Asp Leu Glu Pro Pro Gln Ala Val Gln Pro
Pro 50 55 60Pro Glu Pro Val Val Glu Pro Glu Pro Glu Pro Glu Pro Ile
Pro Glu65 70 75 80Pro Pro Lys Glu Ala Pro Val Val Ile Glu Lys Pro
Lys Pro Lys Pro 85 90 95Lys Pro Lys Pro Lys Pro Val Lys Lys Val Gln
Glu Gln Gln Lys Arg 100 105 110Asp Val Lys Pro Val Glu Ser Arg Pro
Ala Ser Pro Phe Glu Asn Thr 115 120 125Ala Pro Ala Arg Pro Thr Ser
Ser Thr Ala Thr Ala Ala Thr Ser Lys 130 135 140Pro Val Thr Ser Val
Ala Ser Gly Pro Arg Ala Leu Ser Arg Asn Gln145 150 155 160Pro Gln
Tyr Pro Ala Arg Ala Gln Ala Leu Arg Ile Glu Gly Gln Val 165 170
175Lys Val Lys Phe Asp Val Thr Pro Asp Gly Arg Val Asp Asn Val Gln
180 185 190Ile Leu Ser Ala Lys Pro Ala Asn Met Phe Glu Arg Glu Val
Lys Asn 195 200 205Ala Met Arg Arg Trp Arg Tyr Glu Pro Gly Lys Pro
Gly Ser Gly Ile 210 215 220Val Val Asn Ile Leu Phe Lys Ile Asn Gly
Thr Thr Glu Ile Gln225 230 235
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