Note: Descriptions are shown in the official language in which they were submitted.
CA 02610098 2007-11-09
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE _2
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Brevets.
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THAN ONE VOLUME.
THIS IS VOLUME OF _2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02610098 2007-11-09
DESCRIPTION
PRODUCTION OF RECOIMINANT HUMAN HEMOGLOBIN USING PICHIA YEAST
TECHNICAL FIELD OF THE INVENTION
The present invention relates to production of a
recombinant human hemoglobin and an expression vector therefor.
More particularly, the present invention relates to a
production method of a human hemoglobin using a yeast of the
genus Pichia, as well as an expression vector comprising in
io tandem an expression cassette containing a DNA encoding a
human hemoglobin 0-chain and an expression cassette containing
a DNA encoding a human hemoglobin a-chain.
BACKGROUND OF THE INVENTION
Hemoglobin is a protein contained in red blood cells at a
high concentration, and has a function to carry various gas
molecules including oxygen. Structurally, it is a
heterotetramer consisting of two a-chains and two (3-chains.
As a preparation utilizing hemoglobin, a red blood cell
substitute has been developed. There have so far been designed
2o an intramolecular crosslinking type which is a hemoglobin
modification form, a polymerization type, a polymer bond type,
and hemoglobin endoplasmic reticulum in which hemoglobin is
encapsulated in liposome. Particularly, there is a practical
prospect of hemoglobin endoplasmic reticulum as a single
administration oxygen transfusion for critical care, based on
the property data of its shape, hemoglobin purity and the like,
and the biological data of safety, in vivo retention, in vivo
kinetics, oxygen-carrying ability and the like.
On the other hand, a preparation using a human blood-
so derived hemoglobin is associated with the risk of
contamination with unknown virus and the like, and its use for
human body is problematic in terms of safety. However, there
is a possibility of provision of a safe hemoglobin preparation
by a transformed microorganism using the gene recombination
technique.
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Until now, recombinant hemoglobin has been produced
mainly using a system with Escherichia coli as a host cell.
However, this method has problems in that (1) unless an enzyme
(methionine aminopeptidase and the like) is coexpressed with
hemoglobin and the like, addition of redundant first
methionine to the N-terminal occurs, leading to a primary
sequence different from that of a naturally occurring type,
(2) aminolaevulinic acid needs to be added to a culture medium
for the purpose of heme synthesis and the like.
io Two cases have been reported so far as regards the
production of recombinant hemoglobin using Saccharomyces
cerevisiae (see Wagenbach, M. et al., Bio/Technol., 9: 57-61
(1991) and Coghlan, D. et al., Eur. J. Biochem., 207: 931-936
(1992)). In this system, since the first methionine is cleaved
by an enzyme S. cerevisiae itself has, the primary sequence
completely matches with that of a naturally occurring type,
and addition of aminolaevulinic acid for heme synthesis is not
necessary. However, this method is not entirely sufficient due
to the problem of yield and the like, and therefore, an
industrial production of recombinant human hemoglobin has not
been practiced yet.
One of known industrial yeasts other than the genus
Saccharomyces is a yeast of the genus Pichia. This yeast can
grow with methanol as a sole carbon source, and when grown in
methanol, an enzyme necessary for the treatment of methanol
and its metabolism intermediate is disinhibited and expressed.
Production of heterologous protein utilizing the methanol-
utilizing pathway has been studied and applied to the
production of albumin for blood preparations (see e.g., JP-A-
3o 6-22784). The expression amount thereof is known to be very
high, and albumin of a 10 g order can be produced from 1 L of
a medium.
However, there often exist cases where the expression
efficiency of heterologous protein is low due to the
combination (compatibility) of the object heterologous protein
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CA 02610098 2007-11-09
and a host cell. Particularly, in the case of a heterooligomer
protein such as hemoglobin, since many conditions of balanced
expression amounts of respective subunits, oligomer formation
and the like need to be met, the desired high expression
cannot be obtained easily. In fact, a yeast of the genus
Pichia is used for industrial production of human protein in a
very few cases.
An object of the present invention is to provide a means
for producing a safe recombinant protein such as human
io hemoglobin or albumin, globulin and the like, which has
sufficient function and activity similar to those of the
naturally occurring type and is free of viral contamination
and the like, easily in a large amount.
The present inventors have conducted intensive studies in
an attempt to solve the above-mentioned problems and succeeded
in recovering various proteins or peptides such as a human
hemoglobin and the like, particularly a heterooligomer protein
and the like, in a high yield by transforming Pichia pastoris,
which is one of the yeasts of the genus Pichia, with an
2o expression vector containing a DNA encoding plural proteins or
peptide chains, for example, an expression vector containing a
DNA encoding a-chain and 0-chain of human hemoglobin, and
cultivating the obtained transformant in a medium, which
resulted in the completion of the present invention.
SUNbORY OF THE INVENTION
Accordingly, the present invention provides
(1) a production method of a functional recombinant human
hemoglobin, which comprises transforming a yeast of the genus
Pichia with an expression vector containing a DNA encoding a
3o human hemoglobin a-chain, which is under regulation of a
promoter functional in a yeast of the genus Pichia, and an
expression vector containing a DNA encoding a human hemoglobin
(3-chain, which is under regulation of a promoter functional in
a yeast of the genus Pichia, culturing the yeast in a medium,
and recovering the human hemoglobin from the obtained culture;
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CA 02610098 2007-11-09
(2) the method of the above-mentioned (1), wherein the DNA
encoding the human hemoglobin a-chain and the DNA encoding the
human hemoglobin P-chain are on the same vector;
(3) the method of the above-mentioned (2), wherein the DNA
encoding the human hemoglobin R-chain is at the upstream side
in the transcriptional direction;
(4) the method of any of the above-mentioned (1) - (3),
wherein the DNA encoding the human hemoglobin a-chain and the
DNA encoding the human hemoglobin R-chain are under regulation
io of different promoters;
(5) the method of any of the above-mentioned (1) - (4),
wherein the DNA encoding the human hemoglobin a-chain and the
DNA encoding the human hemoglobin R-chain are under regulation
of an alcoholoxidase 1 promoter;
(6) the method of the above-mentioned (5), wherein the
transformed yeast of the genus Pichia is cultivated in a
liquid medium requiring methanol as a single carbon source;
(7) the method of the above-mentioned (6), wherein the culture
is a feeding culture;
(8) the method of any of the above-mentioned (1) - (7),
wherein the yeast of the genus Pichia is Pichia pastoris; and
(9) an expression vector comprising an alcoholoxidase 1
promoter, a DNA encoding the human hemoglobin R-chain, a
terminator functional in a yeast of the genus Pichia, an
alcoholoxidase 1 promoter, a DNA encoding a human hemoglobin
a-chain and a terminator functional in a yeast of the genus
Pichia in this order from the upstream side in the
transcriptional direction.
The present invention also provides
(10) a method of producing an expression vector comprising a
DNA encoding plural proteins, which is under control of a
promoter functional in a yeast of the genus Pichia, wherein
the vector and a DNA encoding one of the plural proteins are
ligated without a dephosphorylation treatment of the vector;
(11) the method of the above-mentioned (10), wherein (i) an
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expression vector containing a DNA encoding one or more
proteins, which is under control of a promoter functional in a
yeast of the genus Pichia, wherein the expression vector is
digested with a restriction enzyme capable of cleavage thereof
other than the inside of the promoter and the DNA and the both
ends produced thereby are not dephosphorylated is provided,
(ii) a DNA encoding other protein, which is under control of a
promoter functional in a yeast of the genus Pichia, wherein
the both ends are complementary to the end of the expression
io vector of the above-mentioned (i) is provided, and (iii) the
expression vector of the above-mentioned (i) and the DNA of
the above-mentioned (ii) are ligated;
(12) a production method of a recombinant protein, which
comprises culturing, in a medium, a yeast of the genus Pichia
transformed with an expression vector obtained by the method
of the above-mentioned (10) or (11), and recovering the plural
proteins from the obtained culture;
(13) the method of the above-mentioned (12), wherein the
plural proteins are polymerized in the culture to form a
functional protein complex; and the like.
Since the human hemoglobin provided by the method of the
present invention is produced using a yeast of the genus
Pichia as a host by the gene recombination technique, it is
free of a risk of contamination with an unknown virus and the
like, which is a problem specific to blood-derived
preparations, and can be used safely for human body and the
like. Using a yeast of the genus Pichia, moreover, a human
hemoglobin can be produced in a large amount, which has
similar function and properties as the naturally occurring
type. Accordingly, administration thereof to a human body etc.
causes less influence such as side effects and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an explanatory drawing of the principle of
connection between hemoglobin a-chain expression cassette and
0-chain expression cassette.
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CA 02610098 2007-11-09
Fig. 2 is an explanatory drawing of the outline of the
procedures for preparing hemoglobin in the Examples.
Fig. 3 is an explanatory drawing of the recombination
method of hemoglobin with Pichia chromosome in the Examples.
DETAILED DESCRIPTION OF THE INVENTION
As a DNA encoding the human hemoglobin a-chain to be
used in the present invention (hereinafter sometimes to be
abbreviated as "Hba"), a DNA containing a base sequence
encoding an amino acid sequence the same or substantially the
io same as the amino acid sequence shown in SEQ ID NO: 2, and as
a DNA encoding a human hemoglobin 0-chain (hereinafter
sometimes to be abbreviated as "Hbp"), a DNA containing a base
sequence encoding an amino acid sequence the same or
substantially the same as the amino acid sequence shown in SEQ
ID NO: 4 can be mentioned.
As the amino acid sequence the same or substantially the
same as the amino acid sequence shown in SEQ ID NO: 2 (or SEQ
ID NO: 4), an amino acid sequence having a homology of not
less than about 80%, preferably not less than about 90%, more
preferably not less than about 95%, particularly preferably
not less than about 97% with the amino acid sequence shown in
SEQ ID NO: 2 (or SEQ ID NO: 4) and the like can be mentioned.
As used herein, by the "homology" is meant the proportion (%)
of the same amino acid and similar amino acid residues
relative to the total overlapping amino acid residues in the
optimal alignment when two amino acid sequences are aligned
using a mathematical algorithm known in the art (preferably,
the algorithm is capable of considering introduction of a gap
into one or both of the sequences for the optimal alignment).
3o The "similar amino acid" means an amino acid similar in the
physicochemical properties. For example, amino acids
classified in the same group such as aromatic amino acids (Phe,
Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar
amino acids (Gln, Asn), basic amino acids (Lys, Arg, His),
acidic amino acids (Glu, Asp), amino acids (Ser, Thr) having a
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CA 02610098 2007-11-09
hydroxyl group, amino acids (Gly, Ala, Ser, Thr, Met) with
small side chain and the like can be mentioned. It is
predicted that substitution with such similar amino acids will
not alter protein phenotypes (namely, preservative amino acid
substitution). Specific examples of preservative amino acid
substitution are well known in the art and are described in
various literatures (see e.g., Bowie et al., Science, 247:
1306-1310 (1990)).
The homology of the amino acid sequence in the present
io specification can be calculated using homology calculation
algorithm NCBI BLAST (National Center for Biotechnology
Information Basic Local Alignment Search Tool) under the
following conditions (expectancy=10; allowing gap;
matrix=BLOSUM62; filtering=OFF). Other algorithms for
determining the homology of the amino acid sequence include,
for example, the algorithm described in Karlin et al., Proc.
Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [this algorithm is
incorporated in the NBLAST and XBLAST program (version 2.0)
(Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))],
the algorithm described in Needleman et al., J. Mol. Biol.,
48: 444-453 (1970) [this algorithm is incorporated in the GAP
program in the GCG software package], the algorithm described
in Myers and Miller, CABIOS, 4: 11-17 (1988) [this algorithm
is incorporated in the ALIGN program (version 2.0) which is a
part of the CGC sequence alignment software package], the
algorithm described in Pearson et al., Proc. Natl. Acad. Sci.
USA, 85: 2444-2448 (1988) [this algorithm is incorporated in
the FASTA program in the GCG software package] and the like,
and they can also be used preferably.
More preferably, an amino acid sequence substantially
same as the amino acid sequence shown in SEQ ID NO: 2 (or SEQ
ID NO: 4) has homology of not less than about 80%, preferably
not less than about 90%, more preferably not less than about
95%, particularly preferably not less than about 97%, to the
amino acid sequence shown in SEQ ID NO: 2 (or SEQ ID NO: 4).
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A protein containing an amino acid sequence substantially
same as the amino acid sequence shown in SEQ ID NO: 2 (or SEQ
ID NO: 4) means a protein containing an amino acid sequence
substantially same as the aforementioned amino acid sequence
shown in SEQ ID NO: 2 (or SEQ ID NO: 4) and having a
substantially equivalent activity to that of the protein
containing the amino acid sequence shown in SEQ ID NO: 2 (or
SEQ ID NO: 4).
The substantially equivalent activity includes, for
io example, the physiological activity of human hemoglobin, for
example, an activity to carry a gas molecule (e.g., oxygen and
the like), and the like. The "substantially equivalent" means
that the activities are qualitatively same. Therefore, the
activity of oxygen-carrying ability and the like is preferably
equivalent (e.g., about 0.5- to about 2-fold). However,
quantitative elements such as the level of the activity,
molecular weight of the protein and the like may be different.
The activity of oxygen-carrying ability and the like
(e.g., autoxidation rate constant, oxygen affinity,
cooperativity and the like) can be measured according to, but
not limited to, a method known per se, for example, the method
described in JP-A-2004-500871 and the like.
In addition, Hba (or HbR) to be used in the present
invention includes, for example, a protein containing (1) an
amino acid sequence wherein one or more (preferably about 1 -
30, more preferably about 1 - 10, particularly preferably 1 -
several (2, 3, 4 or 5)) amino acids are deleted from the amino
acid sequence shown in SEQ ID NO: 2 (or SEQ ID NO: 4), (2) an
amino acid sequence wherein one or more (preferably about 1-
3o 30, more preferably about 1 - 10, particularly preferably 1-
several (2, 3, 4 or 5)) amino acids are added to the amino
acid sequence shown in SEQ ID NO: 2 (or SEQ ID NO: 4), (3) an
amino acid sequence wherein one or more (preferably about 1 -
30, more preferably about 1 - 10, particularly preferably 1 -
several (2, 3, 4 or 5)) amino acids are inserted in the amino
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acid sequence shown in SEQ ID NO: 2 (or SEQ ID NO: 4), (4) an
amino acid sequence wherein one or more (preferably about 1 -
30, more preferably about 1 - 10, particularly preferably 1 -
several (2, 3, 4 or 5)) amino acids are substituted by other
amino acids in the amino acid sequence shown in SEQ ID NO: 2
(or SEQ ID NO: 4), or (5) a combination of these, and the like.
When the amino acid sequence is inserted, deleted or
substituted as mentioned above, the site of the insertion,
deletion or substitution is not particularly limited as long
io as the activity of protein is maintained.
Human hemoglobin in the present invention is used to mean
not only adult hemoglobin (HbAl) but encompass HbA2 wherein a-
chain and S-chain are bonded, fetal hemoglobin (HbF) wherein a-
chain and y-chain are bonded, and the like.
For the production of HbA2 and HbF, therefore, a DNA
encoding S-chain and y-chain instead of 0-chain is used. The
amino acid sequence and base sequence of 8-chain are registered,
for example, in GenBank accession Nos. NP 000510 and NM 000519.
On the other hand, the amino acid sequence and base sequence
of y-chain are registered, for example, in GenBank accession
Nos. NP 000550 and NM 000559.
Examples of the DNA encoding Hba (or Hbp) include human
genomic DNA, cDNA derived from Hba (and/or Hb(3)-producing cells
(e.g., red blood cell and the like) of adult, synthetic DNA
2.5 and the like. The genomic DNA and cDNA encoding Hba (or Hb(3)
can also be directly amplified by Polymerase Chain Reaction
(hereinafter to be abbreviated as "PCR method") and Reverse
Transcriptase-PCR (hereinafter to be abbreviated as "RT-PCR
method"), using a genomic DNA fraction and total RNA or an
3o mRNA fraction prepared from a producing-cell or tissue (e.g.,
blood and the like) thereof as respective templates.
Alternatively, the genomic DNA and cDNA encoding Hba (or Hb(3)
can be respectively cloned by colony or plaque hybridization
method, PCR method and the like, from a genomic DNA library
35 and cDNA library prepared by inserting genomic DNA and total
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RNA or an mRNA fraction prepared from the above-mentioned cell
or tissue into suitable vectors. The vector to be used for the
library may be any such as bacteriophage, plasmid, cosmid,
phagemid and the like.
Examples of the DNA encoding Hba (or HbR) include DNA
containing the base sequence shown in SEQ ID NO: 1 (or SEQ ID
NO: 3), DNA having a base sequence hybridizing with the base
sequence shown in SEQ ID NO: 1 (or SEQ ID NO: 3) under
stringent conditions, and encoding a protein having
lo substantially equivalent activity (e.g., oxygen-carrying
ability and the like) to that of the aforementioned Hba (or
HbR) and the like.
As the DNA capable of hybridizing to the base sequence
shown in SEQ ID NO: 1 (or SEQ ID NO: 3) under stringent
conditions, for example, DNA containing a base sequence having
homology of not less than about 70%, preferably not less than
about 80%, more preferably not less than about 90%,
particularly preferably not less than about 95%, to the base
sequence shown in SEQ ID NO: 1 (or SEQ ID NO: 3) and the like
2o are used.
The homology of the base sequence in the present
specification can be calculated using a homology calculation
algorithm NCBI BLAST (National Center for Biotechnology
Information Basic Local Alignment Search Tool) under the
following conditions (expectancy=l0; allowing gap;
filtering=ON; match score=l; mismatch score=-3). Preferable
examples of other algorithms usable for determining homology
of the base sequence include the above-mentioned homology
calculation algorithms for amino acid sequence.
Hybridization can be performed according to a method
known per se or a method according to the method, for example,
the method described in Molecular Cloning, ver. 2 (J. Sambrook
et al., Cold Spring Harbor Lab. Press, 1989) and the like.
When a commercially available library is used, moreover,
hybridization can be performed according to the method
CA 02610098 2007-11-09
described in the attached instruction manual. Preferably,
hybridization can be performed under stringent conditions.
The high stringent conditions include, for example, a
hybridization reaction at 45 C in 6xSSC (sodium chloride/sodium
citrate), and washing one or more times at 65 C in 0.2xSSC/0.1%
SDS and the like.
Those of ordinary skill in the art can easily adjust to
the desired stringency by appropriately changing the salt
concentration of hybridization solution, temperature of
Io hybridization reaction, probe concentration, length of probe,
number of mismatches, hybridization reaction time, salt
concentration of washing solution, temperature of washing and
the like.
A DNA encoding Hba (or HbR) can be cloned by amplifying
by PCR method using a synthetic DNA primer having a part of
the base sequence encoding Hba (or HbR), or hybridizing DNA
incorporated into a suitable expression vector to
with a labeled DNA fragment or synthetic DNA encoding a part
or full region of Hba (or HbR).
A DNA encoding Hba (or HbR) can be obtained from human
genomic DNA or RNA (cDNA) as mentioned above. It is also
possible to construct a DNA encoding the full-length Hba (or
HbR) by chemically synthesizing a DNA chain or connecting a
synthesized, partly overlapping oligo DNA short chain by PCR
method. The advantage of constructing a full-length DNA by a
combination of chemical synthesis and PCR method is that the
codon to be used can be designed over the full length of the
gene in accordance with the host to be introduced into the
gene. Plural codons encoding the same amino acid are not
uniformly used and the frequency of use varies depending on
the species. In general, a codon contained in a gene highly
expressing in a certain species contains a large amount of
codon frequently used in the species. On the other hand, in
many of the genes with a low expression amount, the presence
of a codon used less frequently often becomes a bottleneck in
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CA 02610098 2007-11-09
the high expression. There are many reports on the examples
wherein, in the expression of a heterologous gene, the
expression amount of the heterologous protein was increased by
substituting the gene sequence with a codon highly frequently
used in the host organism, and therefore, such alteration of
the codon to be used is expected to be effective for
increasing the expression amount of a heterologous gene.
For the above-mentioned reasons, a DNA encoding Hba (or
HbR) can be altered to a codon more suitable for a yeast of the
io genus Pichia into which the DNA is to be introduced (that is,
a codon highly frequently used in the host organism). The
frequency of use of the codon in each yeast of the genus
Pichia is defined by the ratio of use of each codon to all the
genes present on the genomic sequence of the host, for example,
by the number of use per 1000 codons. In addition, the
frequency of use of codon can also be approximately calculated
from plural representative gene sequences in the case of a
biological entity for which the complete genomic sequences
have not been identified. As for the data of frequent use of
codon in yeast of the genus Pichia to be genetically modified,
for example, the genetic code usage frequency database
(http://www.kazusa.or.jp/codon/index.html) disclosed in the
homepage of Kazusa DNA Research Institute can be used.
Alternatively, a document indicating the frequency of use of
codon in each yeast of the genus Pichia may be referred to, or
the data of frequency of use of codon in the yeast of the
genus Pichia to be used may be constructed by oneself. By
referring to the obtained data and the gene sequence to be
introduced, the codon used in the gene sequence, which shows a
low frequency of use in the host yeast, can be substituted to
a codon encoding the same amino acid and showing a high
frequency of use.
Another advantage of constructing a DNA encoding the
full-length Hba (or HbR) by chemically synthesizing a DNA chain,
or connecting a synthesized, partly overlapping oligo DNA
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CA 02610098 2007-11-09
short chain by PCR method is easy construction of an
expression vector thereafter. For example, in the below-
mentioned Examples, an expression cassette of HbR and an
expression cassette of Hba are connected in tandem, utilizing
the complementarity of sticky end produced by digestion with
restriction enzymes BamH I and Bgl II (see Fig. 1. Utilizing
the complementarity of sticky end, multicopy expression
cassette can be repeatedly integrated in tandem into a vector).
Therefore, the DNA encoding Hba and the DNA encoding HbR are
io preferably free of a BamH I recognition site. For example, HbR
cDNA registered as GenBank accession No. NM 000518 in the NCBI
database has a BamH I recognition site (GGATCC) at the 347th -
352nd positions. In the base sequence shown in SEQ ID NO: 3,
the base sequence of the corresponding part (base Nos. 297 -
302) is TGACCC, where a mutation has been introduced to not
contain a BamH I recognition site. In the below-mentioned
Examples, moreover, an EcoR I recognition sequence is added to
both ends of a DNA encoding Hba and a DNA encoding HbR to
construct an expression cassette by inserting a DNA encoding
2o Hba and a DNA encoding HbR between a promoter and a terminator
functional in a yeast of the genus Pichia (see Fig. 2).
Therefore, a DNA encoding Hba and a DNA encoding HbR is
preferably free of a EcoR I recognition site. For example, HbR
cDNA registered as GenBank accession No. NM 000518 in the NCBI
database has an EcoR I recognition site (GAATTC) at the 414th
to 419th positions. In the base sequence shown in SEQ ID NO: 3,
the base sequence of the corresponding part (base Nos. 370 -
375) is GAGTTT, where a mutation has been introduced to not
contain an EcoR I recognition site. When a restriction enzyme
3o recognition site in a protein-coding sequence is to be altered,
it is desirable to substitute a base, thereby to not change
the amino acid sequence of Hba (or HbR) utilizing degeneration
of the codon.
A DNA cloned as mentioned above can be used as it is
depending on the object or used after digestion with a
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CA 02610098 2007-11-09
restriction enzyme or adding a linker. The DNA may have ATG as
a translation initiation codon at the 5' end side thereof and
TAA, TGA or TAG as a translation stop codon at the 3' end side
thereof. These translation initiation codon and translation
stop codon can be added using a suitable synthetic DNA adapter.
An expression vector containing a DNA encoding Hba (or
HbR) can be produced, for example, by ligating a DNA fragment
encoding Hba (or HbR) cloned as mentioned above to a downstream
of a promoter in a suitable expression vector using a
io restriction enzyme and a DNA ligase.
The vector to be used is not particularly limited as long
as it can be maintained stably by autonomous replication in a
fungus body of yeast of the genus Pichia or integration into a
yeast genome. Examples of the autonomously-replicable vector
is include YEp vector, YRp vector, YCp vector and the like. In
addition, examples of the vector to be integrated into a yeast
genome include YIp vector and YRp vector.
Examples of the promoter functional in the yeast of the
genus Pichia include promoters derived from a yeast, such as
20 PH05 promoter, PGK promoter, GAP promoter, ADH promoter
derived from S. cerevisiae and the like, alcohol oxidase (AOX)
1 promoter, AOX2 promoter, dihydroxyacetone synthase promoter,
P40 promoter, ADH promoter, folic acid dehydrogenase promoter
derived from P. pastoris and the like. In addition, the above-
25 mentioned promoter derived from a yeast may be a mutant
promoter modified to further improve the gene expression
efficiency, for example, mutant AOX2 (mAOX2) promoter [Ohi et
al., Mol. Gen. Genet., 243, 489-499 (1994); JP-A-4-299984] and
the like. Preferably, the promoter is a promoter of an enzyme
30 gene necessary for treating methanol or a metabolic
intermediate thereof, in order to use a methanol-metabolizing
system in the yeast of the genus Pichia, such as AOX1 promoter,
mA0X2 promoter and the like, more preferably AOX1 promoter.
The expression vector containing the DNA encoding Hba
35 (or HbR) of the present invention preferably further contains
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CA 02610098 2007-11-09
transcription terminator sequence (terminator) functional in a
yeast of the genus Pichia (e.g., AOX1 terminator etc.),
enhancer sequence, selection marker gene usable for selecting
yeast (auxotrophic gene, for example, HIS4, LEU2, ARG4 and
URA3 gene derived from P. pastoris or S. cerevisiae, and the
like, or antibiotic resistance gene, for example, resistance
gene to cycloheximide, G-418, chloramphenicol, bleomycin,
hygromycin etc., and the like) and the like, and when desired,
may contain replicable unit functional in yeast. For
lo preparation of the vector in a large amount, moreover, the
vector more preferably contains a replicable unit functional
in Escherichia coli and a selection marker gene usable for
selecting Escherichia coli (e.g., resistance gene to
ampicillin and tetracycline etc.).
When the expression vector is of a type incorporated into
a yeast genome, the vector preferably further contains a
sequence homologous to a yeast genome necessary for homologous
recombination. As such homology sequence, the aforementioned
auxotrophic gene sequence can be mentioned. Accordingly, in
one preferable embodiment, the expression vector of the
present invention is one wherein an expression cassette of the
above-mentioned Hba (or HbR) is inserted in an auxotrophic gene
(in the present specification, the "expression cassette" means
a unit enabling gene expression, whose minimal unit is a
protein-coding sequence configured under regulation of a
promoter, with preference given to a unit comprising promoter-
protein-coding region-terminator) (see Fig. 3).
An expression cassette of Hba and an expression cassette
of HbR may be carried on individual vectors or the same vector.
In the former case, two vectors desirably contain different
selection marker genes. However, in a preferable embodiment of
the present invention, the expression cassette of Hba and the
expression cassette of HbR are configured on the same vector.
In this case, the expression cassette of Hba and the
expression cassette of HbR may be configured in any order. For
CA 02610098 2007-11-09
example, the expression cassettes may be configured head-to-
head or tail-to-tail on an opposite chain. Preferably, they
are configured in the same transcriptional direction, more
preferably, the expression cassettes are configured in the
order of the expression cassette of HbR and the expression
cassette of Hba, from the upstream side in the transcriptional
direction. When the DNA encoding Hba and the DNA encoding HbR
are transcribed in the same direction, the transcription may
be performed monocistronically from the same promoter or
io dicistronically from separate promoters. In the case of
monocistronic transcription, the expression vector does not
contain a terminator at the 3' side of the coding sequence in
the upstream. In addition, an internal ribosome entry site
(IRES) is preferably contained at the 5' side of the coding
sequence in the downstream. However, in a preferable
embodiment of the present invention, the DNA encoding Hba and
the DNA encoding HbR are transcribed dicistronically from
separate promoters.
In a particularly preferable embodiment of the present
invention, an expression vector containing AOX1 promoter, DNA
encoding Hbo, terminator functional in a yeast of the genus
Pichia (preferably AOX1 terminator), AOX1 promoter, DNA
encoding Hba and terminator functional in the yeast of the
genus Pichia (preferably AOX1 terminator) in this order from
the upstream side in transcriptional direction is used.
For construction of an expression vector comprising the
above-mentioned expression cassette of HbR and the expression
cassette of Hba ligated in tandem, generally, a site
immediately upstream (when expression cassette of Hba is
contained) of the promoter of the expression vector containing
one of the expression cassettes or a site immediately
downstream (when expression cassette of HbR is contained) of
the terminator thereof is cleaved with a suitable restriction
enzyme (e.g., in Fig. 2, HbR-pA0815 is cleaved with BamH I) to
linearize the vector, and ligated using the other expression
16
CA 02610098 2007-11-09
cassette cleaved by treating with a restriction enzyme
providing the same sticky end (or blunt end) (e.g., in Fig. 2,
expression cassette of Hba is cleaved out from Hba-pA0815 by
double digestion with BamH I and Bgl II) and a DNA ligase. At
this time, in general, a dephosphorylation treatment of the
end (e.g., calf intestine alkaline phosphatase (CIAP)
treatment) is performed to prevent self-ligation on the vector
side. In some cases, the total ligation efficiency can be
enhanced when the dephosphorylation treatment is not applied.
The expression vector obtained as mentioned above can be
introduced into the fungus body of the target yeast of the
genus Pichia using, for example, a known transformation
technique such as competent cell method, protoplast method,
calcium phosphate coprecipitation method, polyethylene glycol
method, lithium method, electroporation method, microinjection
method, liposome fusion method, particle gun method and the
like.
While the yeast of the genus Pichia to be used in the
present invention is not particularly limited, for example, P.
pastoris, Pichia acaciae, Pichia angusta, Pichia anomala,
Pichia capsulata, Pichia ciferrii, Pichia etchellsii, Pichia
fabianii, Pichia farinosa, Pichia guilliermondii, Pichia
inositovora, Pichia jadinii, Pichia methanolica, Pichia
norvegensis, Pichia ofunaensis, Pichia pinus and the like can
be used. Preferred is P. pastoris, particularly, auxotrophic
mutant P. pastoris strain (e.g., P. pastoris GTS115 strain
(HIS4-) [NNRL Y-15851], P. pastoris GS190 strain (ARG4-)
[NNRLY-18014), P. pastoris PPF1 (HIS4-1 URA4-) [NNRL Y-18017]
and the like).
By cultivating the transformed yeast of the genus Pichia
by a method generally used in the art, a functional human
hemoglobin can be produced. The medium to be used needs to
contain at least a carbon source and an inorganic or organic
nitrogen source necessary for the growth of the host cell.
Examples of the carbon source include methanol, glycerol,
17
CA 02610098 2007-11-09
glucose, sucrose, dextran, soluble starch and the like. In
addition, examples of the inorganic or organic nitrogen source
include ammonium salts, nitrate salts, amino acid, corn steep
liquor, peptone, casein, meat extract, yeast extract, soybean
cake, potato extract and the like. When desired, moreover,
other nutrients, for example, inorganic salts such as calcium
chloride, sodium dihydrogenphosphate, magnesium chloride and
the like, vitamins such as biotin and the like, antibiotic and
the like can be added.
Examples of the medium to be used include conventional
natural medium (e.g., YPD medium, YPM medium, YPG medium etc.)
and synthetic medium. As the pH and culture temperature of the
medium, those suitable for the growth of yeast and production
of human hemoglobin are employed. For example, pH of about 5-
about 8, culture temperature of about 20 C - about 30 C are
preferable. In addition, aeration and agitation are performed
as necessary. The culture is generally performed for about 48
- about 120 hr.
For example, when a promoter whose expression is induced
2o by methanol, such as AOX1 promoter, mAOX2 promoter and the
like, is used as a promoter functional in the fungus body of a
yeast of the genus Pichia, a method of liquid aeration-
agitation culture using natural medium controlled to pH about
6.0, which contains glycerol as a carbon source for the growth
of fungus body and methanol as an Hba and HbR expression
inducer is most preferable. When the expression of Hba and HbR
is not preferable for the growth of fungus body, a method
including first increasing the amount of fungus body with a
carbon source other than methanol, and inducing the expression
of Hba and Hbp by addition of methanol is more preferable. In
a culture in a jarfermenter, moreover, a high density culture
method is suitable for the production of human hemoglobin. The
culture may be performed by any of batch culture, feeding
culture and continuous culture, with preference given to
feeding culture method. That is, for a certain period, a
18
CA 02610098 2007-11-09
method including culturing the host fungus body in a medium
(initial medium) containing a carbon energy source suitable
for the growth (e.g., glucose etc.) and/or a nutrient source,
and confining human hemoglobin in the system until completion
of the culture while additionally supplying a substrate
controlling the expression of Hba and HbR (that is, methanol)
to the medium from a certain point in time according to the
situation can be used (see e.g., JP-A-3-83595).
Human hemoglobin produced in the culture can be collected
io in the yeast fungus body by applying the culture after
completion of cultivation to centrifugation and/or filtration,
and subsequently isolated and purified from the fungus body
according to a method known per se. As such method, a method
utilizing the solubility such as salting out, solvent
precipitation and the like; a method mainly utilizing
difference in the molecular weight such as dialysis,
ultrafiltration, gel filtration method, SDS-polyacrylamide gel
electrophoresis and the like; a method utilizing difference in
the electric charge such as ion exchange chromatography and
the like; a method utilizing specific affinity such as
affinity chromatography and the like; a method utilizing
difference in hydrophobicity such as reversed-phase high
performance liquid chromatography and the like; a method
utilizing difference in the isoelectric point such as
isoelectric focusing and the like; and the like can be used.
These methods can be appropriately combined.
Examples of a method for confirming the isolated and
purified human hemoglobin include known Western blotting
method, activity measurement method and the like. In addition,
the structure of the purified hemoglobin can be clarified by
amino acid analysis, N-terminal amino acid sequence, primary
structure analysis and the like. Human hemoglobin obtained in
this way has the same primary structure (that is, removal by
cleavage of first methionine) and higher-order structure as
those of the naturally occurring type, and shows the
19
CA 02610098 2007-11-09
physiological activity of the same quality as the naturally
occurring type.
The recombinant human hemoglobin produced in the present
invention can be administered to the living body, for example,
as a red blood cell substitute. For administration as a red
blood cell substitute, hemoglobin can be administered by
encapsulating in liposome or dispersing in emulsion. The
methods of preparation making and method of administration to
human are described in, for example, JP-A-2004-307404, JP-A-
io 2006-104069, JP-A-2002-161047, JP-A-2001-348341, JP-A-08-
003063, JP-A-08-003062, JP-A-07-017874, JP-A-06-321802, JP-A-
06-072892 and the like.
SEQUENCE LISTING FREE TEXT
SEQ ID NO: 5
primer.
SEQ ID NO: 6
primer.
SEQ ID NO: 7
primer.
SEQ ID NO: 8
primer.
SEQ ID NO: 9
primer.
SEQ ID NO: 10
primer.
SEQ ID NO: 11
primer.
SEQ ID NO: 12
primer.
SEQ ID NO: 13
Hba cDNA having EcoR I site at 5' and 3'-ends.
SEQ ID NO: 14
HbRa cDNA having EcoR I site at 5' and 3'-ends.
SEQ ID NO: 15
primer.
CA 02610098 2007-11-09
SEQ ID NO: 16
primer.
SEQ ID NO: 17
primer.
SEQ ID NO: 18
primer.
SEQ ID NO: 19
primer.
The present invention is explained in more detail in
io the following by referring to Examples, which are mere
exemplifications and do not limit the scope of the present
invention.
EXAMPLES
(1) amplification of hemoglobin gene
Using plasmid pBEX transfected with human hemoglobin gene
(hereinafter referred to as "pBEX-Hb". Available from Tadayuki
Uno (Graduate School and School of Pharmaceutical Sciences,
Osaka University), a coinventor of the present application) as
a template, and Hba-sense primer of SEQ ID NO: 5 and Hba-
2o antisense primer of SEQ ID NO: 6 as synthetic primers of the
DNA sequence of hemoglobin a-chain, and Hb(3-sense primer of SEQ
ID NO: 7 and Hb(3-antisense primer of SEQ ID NO: 8 as synthetic
primers of the DNA sequence of hemoglobin (3-chain, respectively,
mutation of EcoR I restriction enzyme site, which would become
a obstruction of subsequent manipulation, was performed
(QuikChange XL Site-Directed Mutagenesis Kit, Stratagene). As
for mutation reaction conditions, DNA was treated for 30 sec
at 95 C, after which a 12-cycle reaction of denaturation (95 C,
sec), annealing (55 C, 1 min) and extension (68 C, 10 min)
30 was performed. After the reaction, the template plasmid was
digested by Dpn I, and each of obtained pBEX-Hb((x0) and pBEX-
Hb((30) were transfected into XL-10-Gold ultracompetent cells to
perform transformation. The transformants, which were
transfected with the objective plasmids pBEX-Hb(a0) or pBEX-
Hb((3A), were screened in ampicillin-added medium, and the
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plasmids were purified from the obtained transformants
(QIAprep Spin Miniprep Kit, manufactured by QIAGEN). The
plasmids were digested with EcoR I (manufactured by Takara
Shuzo Co., Ltd.), and the mutation was confirmed.
Using the obtained pBEX-Hb(aA) and pBEX-Hb(RA) as
templates, and Hba-sense primer of SEQ ID NO: 9 and Hba-
antisense primer of SEQ ID NO: 10 as synthetic primers of the
DNA sequence of hemoglobin a-chain, and HbR-sense primer of SEQ
ID NO: 11 and HbR-antisense primer of SEQ ID NO: 12 as
lo synthetic primers of the DNA sequence of hemoglobin R-chain,
PCR was performed using a polymerase (KOD-plus-, manufactured
by Toyobo Co., Ltd.). As for PCR reaction conditions, DNA was
treated for 2 min at 94 C, after which a 30-cycle reaction of
denaturation (94 C, 15 sec), annealing (65 C, 30 sec) and
extension (68 C, 1 min) was performed. By this PCR, DNA
fragments, wherein EcoR I restriction enzyme recognition sites
were added to 5' end and 3' end of the DNA sequences encoding
hemoglobin a-chain and R-chain, respectively, were obtained.
The DNA fragments amplified by PCR were purified by phenol
2o extraction, ethanol precipitation, then digested with
restriction enzyme EcoR I (manufactured by Takara Shuzo Co.,
Ltd.). After the restriction enzyme treatment, DNA fragments
were subjected to 2% agarose electrophoresis, and bands
corresponding to each of DNA fragments (DNA fragment encoding
hemoglobin a-chain;Hba, SEQ ID NO: 13 and DNA fragment
encoding hemoglobin R-chain; HbR, SEQ ID NO: 14) were cut out,
and subjected to gel extraction (QIAquick Gel Extraction Kit,
manufactured by QIAGEN).
(2) ligation of hemoglobin gene
E. coli JM109 (manufactured by Takara Shuzo Co., Ltd.)
was transfected with pAO815 (manufactured by Invitrogen), a
shuttle vector for Escherichia coli and yeast, to perform
transformation. The transformant, which was transfected with
pAO815, was screened in ampicillin-added medium, and the
plasmid was purified from the obtained transformant (QIAprep
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CA 02610098 2007-11-09
Spin Miniprep Kit, manufactured by QIAGEN). The plasmid was
digested with EcoR I (manufactured by Takara Shuzo Co., Ltd.),
double-digested with EcoR I and Sal I (manufactured by Takara
Shuzo Co., Ltd.) and double-digested with BamH I and Bgl II
(manufactured by Takara Shuzo Co., Ltd.), by which restriction
enzyme map was constructed, and the plasmid was confirmed to
be an objective plasmid vector.
Escherichia coli, wherein the transfection of pAO815 was
confirmed, was further cultured, and the plasmid was purified
io from grown bacterial bodies (QIAGEN plasmid Maxi Kit,
manufactured by QIAGEN). Purified pA0815 was digested with
EcoR I (manufactured by Takara Shuzo Co., Ltd.), purified by
phenol extraction and ethanol precipitation, after which
dephosphorylation treatment was performed (Alkaline
Phosphatase E. coli C75, manufactured by Takara Shuzo Co.,
Ltd.).
Each of Hba and HbR was ligated to dephosphorylation-
treated pA0815 (DNA Ligation kit Ver. 1, manufactured by
Takara Shuzo Co., Ltd.) to give Hba-pA0815 and HbR-pA0815,
2o respectively. Each of the obtained Hba-pA0815 and HbR-pA0815
was transfected into E. coli JM109 (manufactured by Takara
Shuzo Co., Ltd.) to perform transformation. The transformants,
which were transfected with either the objective plasmids Hba-
pAO815 or HbR-pAO815, were screened in ampicillin-added medium,
and the plasmids were purified from the obtained transformants
(QIAprep Spin Miniprep Kit, QIAGEN manufactured by). The
plasmids were digested with EcoR I (manufactured by Takara
Shuzo Co., Ltd.), and double-digested with BamH I and Bgl II
(manufactured by Takara Shuzo Co., Ltd.), by which restriction
3o enzyme map was constructed, and the plasmids were confirmed to
be Hba-pA0815 and HbR-pA0815.
Furthermore, full sequences of hemoglobin a-chain and
R-chain gene were confirmed by ABI Prism 310 Genetic Analyzer
(Applied Biosystems) using 5' AOX1 sequencing primer of SEQ ID
NO: 15 (manufactured by Invitrogen), Hba-sequence primer 1 of
23
CA 02610098 2007-11-09
SEQ ID NO: 16 and Hba-sequence primer 2 of SEQ ID NO: 17 for
a-chain, and 5' AOX1 sequencing primer of SEQ ID NO: 15
(manufactured by Invitrogen), HbR-sequence primer 1 of SEQ ID
NO: 18 and HbR-sequence primer 2 of SEQ ID NO: 19 for R-chain.
Escherichia coli, wherein the transfection of Hba-pAO815
or HbR-pA0815 were confirmed, was further cultured, and the
plasmids were purified from grown bacterial bodies (QIAGEN
plasmid Maxi Kit, manufactured by QIAGEN). Purified Hba-pA0815
was digested with BamH I and Bgl II (manufactured by Takara
lo Shuzo Co., Ltd.), after which a band corresponding to
Hba expression cassette was cut out, and gel extraction was
performed (QIAquick Gel Extraction Kit, manufactured by
QIAGEN).
On the other hand, purified HbR-pA0815 was digested with
BamH I (manufactured by Takara Shuzo Co., Ltd.) without
performing dephosphorylation treatment, and purified by phenol
extraction and ethanol precipitation.
Hba expression cassette was ligated to HbR-pA0815 (DNA
Ligation kit Ver. 1, manufactured by Takara Shuzo Co., Ltd.),
which was enzyme-digested by BamH I, to give HbR-Hba-pA0815.
Obtained HbR-Hba-pA0815 was transfected into E. coli DH5a
(manufactured by Toyobo Co., Ltd.) to perform transformation.
The transformant, which was transfected with the objective
plasmid HbR-Hba-pA0815, was screened in ampicillin-added medium,
and the plasmid was purified from the obtained transformant
(QIAprep Spin Miniprep Kit, manufactured by QIAGEN). The
plasmid was digested with EcoR I (manufactured by Takara Shuzo
Co., Ltd.), and double-digested with BamH I and Bgl II
(manufactured by Takara Shuzo Co., Ltd.), by which restriction
3o enzyme map was constructed, and the plasmid was confirmed to
be HbR-Hba-pA0815. Fig. 2 shows the procedure for construction
of HbR-Hba-pA0815.
(3) expression of hemoglobin
HbR-Hba-pA0815 was digested with restriction enzyme Sal I,
purified by phenol extraction and ethanol precipitation, and
24
CA 02610098 2007-11-09
subsequently transformed into HIS4 gene locus of Pichia yeast
(GS115 strain) by homologous recombination using an
electroporation apparatus (Gene Pulser II Electroporation
System, manufactured by BIO-RAD) (see Fig. 3). The obtained
transformant was cultured in BMMY liquid medium, and stocked
in glycerol after confirmation of expression of hemoglobin.
(4) purification of hemoglobin
The transformed Pichia yeast was cultured in BMGY liquid
medium for 48 hr, and subsequently in BMMY medium for 96 hr as
io adding 1% methanol every 12 hr. The yeast was precipitated by
centrifugation (6,000 g x 10 min), resuspended in disrupt
buffer, and glass beads (425 - 600 pm) was added, after which
the suspension was stirred intensively to disrupt the yeast.
The disrupted fungus body was separated by centrifugation
at 10,000 g for 30 min, and the supernatant was collected.
This was dialyzed against 10 mM Tris-HC1 buffer (pH 7.5), and
passed through Q-SepharoseTm Fast Flow column (manufactured by
Amersham Biosciences), furthermore, bonded to the Q-SepharoseTm
Fast Flow column by pH adjustment to 8.5, and hemoglobin was
2o eluted by concentration gradient of 0 to 300 mM NaCl. This was
replaced with 200 mM sodium acetate buffer (pH 5.5), bonded to
Blue Sepharose CL-6B column (manufactured by Amersham
Biosciences), and hemoglobin was eluted by concentration
gradient of 0 to 3 M NaCl. Subsequently, this eluate was
dialyzed against 0.5 M ammonium sulfate/100 mM sodium
phosphate buffer (pH 7.0), bonded to HiTrap Butyl FF column
(manufactured by Amersham Biosciences), and hemoglobin was
eluted with 70% ethanol. Finally, CO was blown into the eluate
and the eluate was preserved at 4 C.
(5) confirmation of hemoglobin
The final sample was subjected to SDS polyacrylamide gel
electrophoresis, and a single band of predicted molecular
weight, about 15,000, was detected. Also, N-terminal amino
acid analysis was performed, and it showed that the band
completely corresponded with the amino acid sequence of human
CA 02610098 2007-11-09
=
blood-derived hemoglobin. Furthermore, molecular weights of a-
chain and (3-chain were estimated by MALDI TOF-MS, 15,122 and
15,866, respectively, corresponding with calculated weights.
The production method of the present invention is free of
a risk of contamination with virus and the like. Therefore, it
is extremely useful in that it can conveniently provide human
hemoglobin in large amounts, which can be safely administered
to the living body for medical purposes.
While the present invention has been described with
lo emphasis on preferred embodiments, it is obvious to those
skilled in the art that the preferred embodiments can be
modified. The present invention intends that the present
invention can be embodied by methods other than those described
in detail in the present specification. Accordingly, the present
invention encompasses all modifications encompassed in the gist
and scope of the appended "CLAIMS."
In addition, the contents disclosed in any publication
cited herein, including patents and patent applications, are
hereby incorporated in their entireties by reference, to the
2o extent that they have been disclosed herein.
26
CA 02610098 2007-11-09
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