Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF RECOMBINANT MONELLIN USING
METHYLOTROPHIC YEAST EXPRESSION SYSTEM
This application claims the benefit of priority under 35 U.S.C. ~119(e) to __
U.S. provisional application Serial No. 60/114,529 to Lingxun Duan, filed
December 31, 1998, and entitled PRODUCTION OF RECOMBINANT
MONELLIN USING METHYLOTROPHIC YEAST EXPRESSION SYSTEM.
1. FIELD OF THE INVENTION
The present invention relates to a single-chain monellin-like protein which is
stable and which is at least 100-fold sweet as compared to sucrose on the
weight
basis. The present invention also relates to a nucleic acid encoding said
monellin-
like protein. Preferably, the nucleic acid further comprises a promoter and a
signal
sequence for directing expression and secretion of the encoded monellin-like
protein in the methylotrophic yeast Pichia pastoris. The present invention
further
relates to a recombinant Pichicr pastoris cell containing the nucleic acid
encoding
the monellin-like protein, a process for producing the monellin-like protein
from
the recombinant Pichica pastoris and product of the process.
2. BACKGROUND ART
2.1. MONELLIN
Monellin belongs to a family of intensely sweet proteins derived from
tropical plants (Dansby, Nata~re Biotechnology, 1997, 15:419-420). Monellin is
about 3,000-fold sweet as compared to sucrose. Other similar proteins include
thaumatin, miraculin, mabinlin, pentadin and aspartame (Id.) Monellin was
first
isolated from the West African Plant Dioscoreophyllum comminisii (U.S. Patent
Nos. 3,878, I 84 and 3,998,798; Morris and Cagan, Biochim. Biophys. Acta,
1972,
261:114-122). The amino acid sequence, the three-dimensional structure and
various physical and chemical properties of monellin have been characterized
(Ogata, et al., Nature, 1987, 328:739-742; Morris et al., J. Biol. Chem.,
1973,
248:534-539; Cagan, Science, 1973, 181:32-35; Bohak and Li, Biochim. Biophys.
Acta, 1976, 427:153-170; Hudson and Beeman, Biochem. Biophys. Res. Comm.,
1976, 71:212-220; Van der Wel and Loeve, FEBS Lett., 1973, 29:181-183; and
Frank and Zuber, HoppeSeyler's Z Physiol. Chem., 1976, 357:585-592).
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U.S. Patent No. 4,300,576 discloses smoking articles containing thaumatin
or monellin. U.S. Patent No. 4,562,076 discloses chewing gum with coating of
thaumatin or monellin. U.S. Patent No. 4,412,984 discloses flavor potentiated
oral
compositions containing thaumatin or monellin. However, despite its potential
as
low-calorie sweeteners, wide commercial application of monellin is hampered by
concerns over its poor stability to heat and pH, lack of access to sources of
supply
of the plant and uncertainty in the regulatory climate for food additives
(Dansby,
Natttre Biotechnology, 1997, 15:419-420).
In 1989, Sung-Hou Kim's group reported production of single-chain
monellin in E. coli by genetic engineering (Kim et al., Protein Eng., 1989,
2:571-
575). The purified single-chain monellin was found to be more heat-stable and
tolerant to a wide pH range, but retained the intensity of sweetness. Several
aspects of this invention have been the subject of certain U.S. patents. For
example, U.S. Patent No. 5,234,834 discloses constructs for expression of
single-
I S chain monellin in plant cells. U.S. Patent No. 5,487,923 discloses a sweet
proteinaceous compound of the formula B-C-A, wherein B represents a peptide
portion at least 90% homologous to residues I -46 of the B chain of native
monellin
and modified only by conservative substitutions; C is a covalent bond or is a
hydrophilic, physiologically acceptable covalent linker capable of providing a
spacing length equivalent to a peptide of 1-10 amino acids selected so as to
reside
on the external portion of the molecule and not to disturb the native
conformation;
and A represents a peptide at least 90% homologous to residues 6-45 of the A
chain of native monellin and modified only by conservative substitution. U.S.
Patent No. 5,487,983 discloses an expression system for making the single-
chain
monellin disclosed in U.S. Patent No. 5,487,923. U.S. Patent No. 5,670,339
discloses DNA encoding the single-chain monellin disclosed in U.S. Patent No.
5,487,923. U.S. Patent No. 5,672,372 discloses methods for sweetening a food
composition with the single-chain monellin disclosed in U.S. Patent No.
5,487,923.
U.S. Patent No. 5,264,558 discloses a single-chain monellin protein that is,
in a
standard taste test, at least 50 times that of sucrose on a weight basis.
Recently, Kondo et al., Nature Biotechnology, 1997, 15:453-457 discloses
heterologous expression of a single-chain monellin protein in the yeast
Candida
utilis intracellularly. It reports that monellin was produced at a high level,
accounting for >50% of the soluble protein.
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2.2. EXPRESSION OF HETEROLOGOUS PROTEINS
IN PICHIA PASTORIS
The methylotrophic yeast Pichia pastoris has been used as a protein
expression system. Several aspects of this expression system have been the
subject
of certain U.S. patents. For example, U.S. Patent No. 4,837,148 discloses
autonomous replication sequences for Pichia pastorzs. U.S. Patent No.
4,855,231
discloses regulatory region for heterologous gene expression in Pichia
pastoris
cells. U.S. Patent No. 4,882,279 discloses site selective genomic modification
of
Pichia pastoris. U.S. Patent No. 4,929,555 discloses a method for making whole
cells of Pichia pastoris competent for transformation. U.S. Patent No.
5,122,465
discloses a process for generating a selectable phenotype in strains of Pichia
pczstor'is. U.S. Patent No. 5,324,639 discloses production of insulin-like
growth
factor-1 in methylotrophic cells, including Pichia pastoris cells.
A number of signal sequences have been used to direct secretion of
heterologous proteins expressed in Pichia pastoris cells. Examples of such
signal
sequences include, but are not limited to, the signal sequence of Pichia
pastoris
acid phosphatase, the signal sequence of Aspergillzzs gigafzteus alpha-Sarcin
(Martinez-Ruiz et al., Protein E.ipr. Pzzrif., 1998, 12(3):315-22; Abdulaev et
al.,
Protein Erpr. Pur'if., 1997, 10(1):61-9; Kotake et al., J. Lipid Res., 1996,
37(3):599-605), the signal sequence of alpha-N-Acetylgalactosaminidase
(alphaNAGAL, EC 3.2.1.49) (Zhu et al., Arch. Biochem. Biophys., 1998,
352(1):1-8), the signal peptide of the OmpA protein (Heim et al., Biochim.
Bioply~s. Acta., 1998, 1396(3):306-19), the signal sequence of the mouse
alpha-factor signal (cCel l ) or the native signal sequence of pepper
endo-beta-1,4-glucanases (Ferrarese et al., FEBS Lett., 1998, 422(1):23-6),
signal
peptide of laccase isolated from the ligninolytic fungus Trametes (Jonsson et
al.,
Curr. Genet., 1997, 32(6):425-30), signal peptide of murine lysosomal acid
alpha-mannosidase (Merkle et al., Biochim. Biophys. Acta., 1997, 1336(2):132-
46),
signal peptide of the porcine inhibitor of carbonic anhydrase (Wuebbens et
al.,
Biochemistry, 1997, 36 14 :4327-36), signal sequence of Aspergillus awamori
glucoamylase (Fierobe et al., Protein Expr. Purif., 1997, x:159-70), signal
sequence of mouse major urinary protein (Ferrari et al., FEBS Lett., 1997,
401 1 :73-7), signal sequence of phol (Skory et al., Curr. Genet., 1996,
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30(5):417-22), signal sequence of rabbit angiotensin-converting enzyme (ACE)
(Sadhukhan et al., J. Biol. Chem., 1996, 271(31):18310-3), prepeptide sequence
of
Pichia pastoris aspartic proteinase (Tsujikawa et al., Yeast, 1996, 12(6):541-
53),
signal sequence of Pichia pastoris PRC1 (Ohi et al., Yeast, 1996, 12(1):31-
40), the
signal sequence of a bacterial thermostable alpha amylase and SUC2 gene signal
sequence from Saccharomyces cerevisiae (Paifer et al., Yeast, 1994,
10(11):1415-9)
and the signal sequence of Saccharomyces cerevisiae mating pheromone a-factor
(Fidler et al., J. Mol. Endocrinol., 1998, 21 3 :327-336).
Although the methylotrophic yeast Piclria pastoris has been used
successfully for the production of various heterologous proteins, U.S. Patent
No.
5,324,639 discloses that at the present level of understanding of
methylotrophic
yeast expression systems, it is unpredictable whether a given gene can be
expressed
to an appreciable level in such yeast or whether the yeast host will tolerate
the
presence of the recombinant gene product in its cells. U.S. Patent No.
5,324,639
IS further discloses that it is especially difficult to foresee if a
particular protein will
be secreted by the methylotrophic yeast host, and if it is, at what
efficiency. For
example, Vollmer et al., J. Immunol. Methods, 1996, 199( I ):47-54, reports
that
when the 323 amino acid residues of the human sIL-6R are inserted into an
expression/secretion vector suitable for the methylotrophic yeast Pichia
pastoris, no
detectable expression and secretion of the recombinant protein was obtained.
Up to
date, monellin has not been expressed and secreted using the Pichia pastoris
expression system.
Given the great interest in the commercial application of monellin, there is a
great need for a more efficient method for producing stable monellin which
still
retains its native sweet intensity and which simplify down stream purification
procedures. The present invention addresses these and other needs in the art.
Citation of references hereinabove shall not be construed as an admission that
such
references are prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention relates to an isolated nucleic acid comprising a
nucleotide sequence encoding a chimeric protein, said chimeric protein
comprises,
from N-terminus to C-terminus: a) a first peptidyl fragment consisting of an
amino
acid sequence that has at least 40% identity to residues 1-50 of the B chain
of
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native monellin, in which the percentage identity is determined over an amino
acid
sequence of identical size to the B chain of native monellin; b) a peptidyl
bond, or
a second peptidyl fragment consisting of 1-12 amino acids; and c) a third
peptidyl
fragment consisting of an amino acid sequence that has at least 40% identity
to
5 residues 1-45 of the A chain of native monellin, in which the percentage
identity is
determined over an amino acid sequence of identical size to the A chain of
native
monellin, wherein said chimeric protein is stable and a given amount of said
chimeric protein is at least 100-fold sweet as compared to the identical
amount of
sucrose, and within said nucleic acid, codons which are preferably used by
yeast
cells are used. Preferably, the isolated nucleic acid further encodes a
promoter
which is capable of directing protein expression in Pichia pastoris and/or an
amino
acid sequence which is capable of directing secretion of the encoded chimeric
protein from Pichia pastof-is.
The present invention also relates to a recombinant Piclzicr pastoris cell
containing the above nucleic acids. The present invention further relates to a
process for producing a chimeric protein comprising growing a recombinant
Pichia
pastoris cell containing the above nucleic acid such that the encoded chimeric
protein is expressed and secreted by the cell, and recovering the expressed
and
secreted chimeric protein. Finally, the present invention relates to products
of the
above processes.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequence of a recombinant single-chain
monellin protein and the DNA sequence encoding the recombinant single-chain
monellin protein. Amino acid residues 1-50 corresponds to the amino acid
residues
1-SO of the B chain of native monellin; amino acid residue 51 is Glycine as
the
linker; and amino acid residues 52-96 corresponds to the amino acid residues 1-
45
of the A chain of native monellin.
FIG. 2 shows the DNA sequence of the oligos which were used for
synthesis of the recombinant single-chain monellin protein.
FIG. 3 shows the location of each of DNA oligo in the synthesized monellin
DNA and its enzymatic digestion sites.
FIG.4 shows the restriction map of recombinant monellin protein expression
vector pGWYSI.
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FIGS shows the construction of pGWYSI.
FIG.6 shows the SDS-PAGE analysis of the secreted recombinant monellin
protein isolated from the culture medium.
FIG.7 shows the steps for purifying the secreted recombinant monellin
protein.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a nucleic acid encoding a single-chain
monellin-like protein which is stable and which is at least 100-fold sweet as
IO compared to sucrose on the weight basis. Preferably, the nucleic acid
further
comprises a promoter and a signal sequence for directing expression and
secretion
of the encoded monellin-like protein in the methylotrophic yeast Pichia
pcrstoris.
The present invention also provides a recombinant Pichia pastoris cell
containing
the nucleic acid encoding the monellin-like protein, a process for producing
the
monellin-like protein from the recombinant Pichia pastoris and product of the
process.
For clarity of disclosure, and not by way of limitation, the detailed
description of the invention is divided into the subsections which follow.
5.1. NUCLEIC ACIDS ENCODING
THE SINGLE-CHAIN MONELLIN PROTEINS
The present invention provides an isolated nucleic acid comprising a
nucleotide sequence encoding a chimeric protein, said chimeric protein
comprises,
from N-terminus to C-terminus: a) a first peptidyl fragment consisting of an
amino
acid sequence that has at least 40% identity to residues 1-50 of the B chain
of
native monellin, in which the percentage identity is determined over an amino
acid
sequence of identical size to the B chain of native monellin; b) a peptidyl
bond, or
a second peptidyl fragment consisting of 1-12 amino acids; and c) a third
peptidyl
fragment consisting of an amino acid sequence that has at least 40% identity
to
residues 1-45 of the A chain of native monellin, in which the percentage
identity is
determined over an amino acid sequence of identical size to the A chain of
native
monellin, wherein said chimeric protein is stable and a given amount of said
chimeric protein is at least 100-fold sweet as compared to the identical
amount of
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sucrose, and within said nucleic acid, codons which are preferably used by
yeast
cells are used. __
In a specific embodiment, the present invention provides an isolated nucleic
acid comprising a nucleotide sequence encoding the chimeric protein wherein
the
first peptidyl fragment consists of an amino acid sequence that has at least
60%
identity to the B chain of native monellin. Preferably, the first peptidyl
fragment
consists of an amino acid sequence that has at least 90% identity to the B
chain of
native monellin. More preferably, the first peptidyl fragment consists of the
amino
acid residues 1-SO of the B chain of native monellin.
In another specific embodiment, the present invention provides an isolated
nucleic acid comprising a nucleotide sequence encoding the chimeric protein
wherein the second peptidyl fragment consists of the amino acid sequence Gly-
Gly-
Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser (SEQ ID NO: l ). Preferably, the
second
peptidyl fragment consists of the amino acid sequence Gly-Gly-Gly-Ser (SEQ ID
N0:2). More preferably, the second peptidyl fragment consists of amino acid
residue Gly.
In still another specific embodiment, the present invention provides an
isolated nucleic acid comprising a nucleotide sequence encoding the chimeric
protein wherein the third peptidyl fragment consists of an amino acid sequence
that
has at least 60% identity to the A chain of native monellin. Preferably, the
third
peptidyl fragment consists of an amino acid sequence that has at least 90%
identity
to the A chain of native monellin. More preferably, the third peptidyl
fragment
consists of the amino acid residues I-45 of the A chain of native monellin.
In a preferred embodiment, the present invention provides an isolated
nucleic acid comprising a nucleotide sequence encoding the chimeric protein
wherein the first peptidyl fragment consists of the amino acid residues 1-SO
of the
B chain of native monellin, the second peptidyl fragment consists of the amino
acid
residue Gly and the third peptidyl fragment consists of the amino acid
residues 1-
45 of the A chain of native monellin.
In a specific embodiment, the present invention provides an isolated nucleic
acid comprising a nucleotide sequence encoding the chimeric protein which is
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capable of being immunoreactively bound by an anti-monellin or an anti-
thaumatin
antibody. __
In another specific embodiment, the present invention provides an isolated
nucleic acid comprising a nucleotide sequence encoding the chimeric protein
S wherein the chimeric protein further comprises an amino acid sequence which
is
capable of directing secretion of said chimeric protein from Pichia pastoris.
Preferably, the secretion-directing sequence is an endogenous signal sequence
of
Pichiu pastoris. More preferably, the endogenous signal sequence is selected
from
the group consisting of the signal sequence of Pichia pastoris acid
phosphatase,
Pichia pastor-is aspartic proteinase and Pichia pastoris carboxypeptidase Y
encoded
by Pichia pastoris PRC1. Alternatively, the secretion-directing sequence is a
yeast
signal sequence, wherein said yeast is not Pichia pastoris. Preferably, the
yeast
signal sequence is a signal sequence from Saccharoyvces cerevisiae. More
preferably, the Saccharomvces cerevisiae signal sequence is selected from the
group
consisting of the signal sequence of Saccharomvces cerevisiae SUC 2 and
Succharoyvces cerevisiae mating pheromone a-factor. Most preferably, the
Saccharomyces cerevisiae signal sequence is the signal sequence of
Saccharomyces cerevisiae mating pheromone a-factor. Examples of other
secretion-directing sequences that can be used in the present invention
include, but
are not limited to, the signal sequence of Aspergillus giganteus alpha-Sarcin,
alpha-N-Acetylgalactosaminidase, OmpA protein, the mouse alpha-factor (cCel l
),
the pepper endo-beta-1,4-glucanases, the laccase isolated from the
ligninolytic
fungus Tr-ametes, murine lysosomal acid alpha-mannosidase, the porcine
inhibitor
of carbonic anhydrase, Aspergillzcs awamori glucoamylase, mouse major urinary
protein, phol, rabbit angiotensin-converting enzyme (ACE), and the bacterial
thermostable alpha amylase.
In a specific embodiment, the present invention provides an isolated nucleic
acid comprising a nucleotide sequence encoding the chimeric protein which
nucleic
acid is a DNA. In another specific embodiment, the present invention provides
an
isolated nucleic acid which is hybridizable to the DNA sequence encoding the
chimeric protein. In still another specific embodiment, the present invention
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provides an isolated nucleic acid comprising a nucleotide sequence
complementary
to the nucleotide sequence encoding the chimeric protein. __
In a specific embodiment, the present invention provides a DNA encoding
the chimeric protein which DNA further comprises a promoter which is capable
of
directing protein expression in Pichia pastoris. Preferably, the promoter is
an
endogenous promoter of Pichia pastoris. More preferably, the endogenous
promoter is the promoter of Pichia pastoris glyceraldehyde-3-phosphate
dehydrogenase (GAP). Alternatively, although not preferred, promoters of
methanol responsive genes in methylotrophic yeast can also be used. Examples
of
such methanol responsive promoters include, but are not limited to, the
promoter
for the primary alcohol oxidase gene from Pichia pastoris AOXI, the promoter
for
the secondary alcohol oxidase gene from Piclzia pastoris AOX2, the promoter
for
the dihydroxyacetone synthase gene from Pichia pastoris (DAS), the promoter
for
the P40 gene from Pichia pastoris, the promoter for the catalase gene from
Piclzia
pastoris, and the like (see U.S. Patent No. 5,324,639).
In another specific embodiment, the present invention provides a DNA
encoding the chimeric protein which DNA further includes sequences allowing
for
its replication and selection in bacteria. In this way, large quantities of
the DNA
fragment can be produced by replication in bacteria.
In a preferred embodiment, the present invention provides a DNA encoding
the chimeric protein, wherein within the encoded chimeric protein, the first
peptidyl
fragment consists of the amino acid residues 1-50 of the B chain of native
monellin, the second peptidyl fragment consists of the amino acid residue Gly
and
the third peptidyl fragment consists of the amino acid residues 1-45 of the A
chain
of native monellin, and said DNA further comprises the promoter of Pichia
pastoris GAP and the signal sequence of Saccharomyces cerevisiae mating
pheromone a-factor.
In another preferred embodiment, the present invention provides a DNA
encoding the chimeric protein, wherein the codons which are preferably used by
Pichia pastoris cells are used.
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In a most preferred embodiment, the present invention provides a DNA
encoding the chimeric protein wherein the DNA molecule comprises nucleotide __
sequence as depicted in Figure 1 or the DNA vector as depicted in Figure 4.
The nucleic acid comprising a nucleotide sequence encoding the chimeric
S protein disclosed herein, or any fragments, analogues or derivatives
thereof, can be
obtained by any methods) known in the art. The nucleic acid may be chemically
synthesized entirely. Alternatively, the nucleic acid encoding each fragment
of the
chimeric protein, i.e., the first, second or third peptidyl fragment, may be
obtained
by molecular cloning or may be purified from the desired cells. The nucleic
acid
10 encoding each fragment of the chimeric protein may then be chemically or
enzymatically ligated together to form the nucleic acid comprising a
nucleotide
sequence encoding the chimeric protein disclosed herein, or any fragments,
analogues or derivatives thereof.
Any Dioscoreophyllun r cornntinisii cell potentially can serve as the nucleic
1 S acid source for the isolation of the nucleic acids encoding monellin.
Alternatively,
the nucleic acids encoding monellin can be designed and synthesized according
to
the amino acid sequence of the native monellin depicted in Figure 1 (see also
U.S.
Patent No. 5,478,923).
The DNA may be obtained by standard procedures known in the art from
cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or
by the cloning of genomic DNA, or fragments thereof, purified from the desired
cell (See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press,
Ltd., Oxford, U.K. Vol. I, IL) Clones derived from genomic DNA may contain
regulatory and intron DNA regions in addition to coding regions; clones
derived
from cDNA will contain only exon sequences. Whatever the source, the gene
should be molecularly cloned into a suitable vector for propagation of the
gene.
In the molecular cloning of the gene from cDNA, cDNA is generated from
totally cellular RNA or mRNA by methods that are well known in the art. The
gene may also be obtained from genomic DNA, where DNA fragments are
generated (e.g., using restriction enzymes or by mechanical shearing), some of
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which will encode the desired gene. The linear DNA fragments can then be
separated according to size by standard techniques, including but not limited
to, __
agarose and polyacrylamide gel electrophoresis and column chromatography.
Once a nucleic acid comprising a nucleotide sequence encoding the chimeric
protein disclosed herein, or any fragments, analogues or derivatives thereof,
has
been obtained, its identity can be confirmed by nucleic acid sequencing (by
any
method well known in the art) and comparison to the known sequences. DNA
sequence analysis can be performed by any techniques known in the art,
including
but not limited to the method of Maxam and Gilbert (Maxam and Gilbert, 1980,
Meth. Enzymol., 65:499-560), the Sanger dideoxy method (Sanger et al., 1977,
Proc. Natl. Acad. Sci. U.S.A., 74:5463), the use of T7 DNA polymerase (Tabor
and
Richardson, U.S. Patent No. 4,795,699), use of an automated DNA sequenator
(e.g., Applied Biosystems, Foster City, CA) or the method described in PCT
Publication WO 97/15690.
Nucleic acids which are hybridizable to a nucleic acid comprising a
nucleotide sequence encoding the chimeric protein disclosed herein, or any
fragments, analogues or derivatives thereof, can be isolated, by nucleic acid
hybridization under conditions of low, high, or moderate stringency (See also
Shilo
and Weinberg, 1981, Proc. Natl. Acad. Sci. USA, 78:6789-6792).
As used herein, "stable" means that a claimed single-chain monellin
chimeric protein retains at least 70% of its sweet intensity after the protein
has
been placed at about 4°C for at least 6 months, or at about 60°C
for at least 2.5
hours, or at about 100°C for at least 5 minutes. In addition, "stable"
means that a
claimed single-chain monell'in chimeric protein retains at least 70% of its
sweet
intensity after the protein has been placed at a pH raging from about 2.0 to
about
11.0 for at least 6 hours.
Sweetness of the claimed single-chain monellin chimeric protein can be
assessed using an ordinary taste test that is known in the art. For example,
comparison to the sweetness of sucrose can be made by suitable dilutions on a
weight basis (see also U.S. Patent No. 5,478,923).
The preferred codon usage by yeast cells can be determined by methods
known in the art, e.g., methods disclosed in Sharp et al., Nucleic Acids Res.,
1986,
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14(13):5125-43 and in Li and Luo, J. Theor. Biol., 1996, 181 2 :111-24.
According to Sharp, important characteristics of the preferred codon in yeast
__
include a higher correlation with tRNA abundance, a greater degree of third
base
pyrimidine bias, and a lesser tendency to the A+T bas pairs. Li and Luo
discloses
a method of classifying and predicting the gene expression level in E. coli
and
yeast cells which is called the Self Consistent Information Clustering (SCIC).
Using the modified Codon Adaption Index (CAI) values, Li and Luo have
accomplished the linear regression analysis on the relation between base
composition, base correlation and gene expression level in Escherichia coli
and
yeast. Li and Luo also proposed the assumption of Expression-Enhancing-Network
Site (EENS), the existence of which can be demonstrated by the linear
equations
between gene expression and base correlations in a codon, in adjacent codons
and
in non-adjacent codons. In addition, the codons that have been successfully
used
for expressing heterologous proteins in Pichia pastoris cells can be used.
Examples
of such codons can be found in U.S. Patent No. 4,837,148; U.S. Patent No.
4,855,231; U.S. Patent No. 4,882,279; U.S. Patent No. 4,929,555; U.S. Patent
No.
5,122,465; U.S. Patent No. 5,324,639; Martinez-Ruiz et al., Protein E.xpr.
Purif.,
1998, 12(3):315-22; Abdulaev et al., Protein Expr. Prrrif., 1997, 10(1):61-9;
Kotake et al., J. Lipid Res., 1996, 30:599-605; Zhu et al., Arch. Biochenz
Bioplzys., 1998, 352 1 :1-8; Heim et al., Biochinr. Biophys. Acta., 1998,
1396 3 :306-19; Ferrarese et al., FEBSLett., 1998, 422(1):23-6; Jonsson et
al.,
Carrr. Genet., 1997, 32 6 :425-30; Merkle et al., Biochim. Biophl's. Acta.,
1997,
1336(2):132-46; Wuebbens et al., Biochemistry, 1997, 36y14):4327-36; Fierobe
et
al., Protein E.xpr. Purif., 1997, 9~2~:159-70; Ferrari et al., FEBS Lett.,
1997,
401 ( 1 ): 73-7; Skory et al., Curr. Genet. , 1996, 30 5 :417-22; Sadhukhan et
al., J.
Biol. Chern., 1996, 271 31 :18310-3; Tsujikawa et al., Yeast, 1996, 12 6 :541-
53;
Ohi et al., Yeast, 1996, 12(1):31-40; Paifer et al., Yeast, 1994, 10 11 :1415-
9;
Fidler et al., J. Mol. Endocrinol., 1998, 21 3 :327-336; and Brocca et al.,
Protein
Sci., 1998, x:1415-22.
Whether a chimeric protein is capable of being immunoreactively bound by
an anti-monellin or an anti-thaumatin antibody can be determined by methods
known in the art. The examples of anti-monellin or an anti-thaumatin
antibodies
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13
that can be used in the present invention include, but are not limited to, the
antibodies disclosed in Slootstra et al., Chem. Senses, 1995, 20 5 :535-43; __
Antonenko and Zanetti , Life Sci., 1994, 55(15):1187-92; Bodani et al.,
Hybridoma,
1993, 12(2):177-83; Mandal et al., Hybridorna, 1991, 10(4):459-66 and
Haimovich,
Isf-. J. Med. Sci., 1975, 11 ( 11 ):1183.
5.2. PRODUCTION OF MONELLIN PROTEINS
FROM RECOMBINANT PICHIA PASTOR1S CELLS
In a specific embodiment, the present invention provides a recombinant
Pichicr pcrstoris cell containing the nucleic acid which encodes a chimeric
protein,
said chimeric protein comprises, from N-terminus to C-terminus: a) a first
peptidyl
fragment consisting of an amino acid sequence that has at least 40% identity
to
residues 1-50 of the B chain of native monellin, in which the percentage
identity is
determined over an amino acid sequence of identical size to the B chain of
native
monellin; b) a peptidyl bond, or a second peptidyl fragment consisting of 1-12
amino acids; and c) a third peptidyl fragment consisting of an amino acid
sequence
that has at least 40% identity to residues 1-45 of the A chain of native
monellin, in
which the percentage identity is determined over an amino acid sequence of
identical size to the A chain of native monellin, wherein said chimeric
protein is
stable and a given amount of said chimeric protein is at least 100-fold sweet
as
compared to the identical amount of sucrose, and within said nucleic acid,
codons
which are preferably used by yeast cells are used. Preferably, the recombinant
Pichia pastoris cell contains a DNA molecule comprises nucleotide sequence as
depicted in Figure 1 or a DNA vector as depicted in Figure 4. Recombinant
Pichicr pcrstoris cells containing the nucleic acids disclosed in Section 4.1.
are also
provided.
Methods for transforming methylotrophic yeast, such as Pichia pastoris, as
well as methods applicable for culturing methylotrophic yeast cells containing
in
their genome a gene encoding a heterologous protein, are known generally in
the
art. Preferably, the transformation, positive transformant selection and
culturing
methods disclosed in U.S. Patent No. 4,837,148; U.S. Patent No. 4,855,231;
U.S.
Patent No. 4,882,279; U.S. Patent No. 4,929,555; U.S. Patent No. 5,122,465;
U.S.
Patent No. 5,324,639 can be used in the present invention.
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14
In another specific embodiment, the present invention provides a process for
producing a monellin chimeric protein comprising growing a recombinant Pichia
__
pastoris cell containing the nucleic acid disclosed in Section 4.1. such that
the
encoded chimeric protein is expressed and secreted by the cell, and recovering
the
expressed and secreted chimeric protein. Preferably, the recombinant Pichia
pastoris cell containing a DNA molecule comprises nucleotide sequence as
depicted
in Figure 1 or a DNA vector as depicted in Figure 4 is used.
Any suitable fermentation process in the art can be used in the present
process. For large-scale production of recombinant DNA-based products driven
by
a GAP promoter in methylotrophic yeast such as Pichia pastoris, a three-stage,
high cell-density fed batch fermentation system is preferably employed. In the
first
or growth stage of this fermentation system, the expression host Pichia
pcrstoris
cells are cultured in defined minimal medium such as BMGY (Buffered Minimal
Glycerol-complex medium) with an excess of a non-inducing carbon source (e.g.,
glycerol). When the expression host PiclTia pastoris cells are grown on such
carbon sources, heterologous gene expression is repressed, which allows the
generation of cell mass in the absence of heterologous protein expression.
During
this growth stage, it is also preferred that the pH of the medium be
maintained at
about 5. Next, the expression host Pichia pastoris cells are grown on limited
non-
inducing carbon source for a short period of time to further increase the cell
mass
and to depress the glucose responsive promoter. The pH of the medium during
this
limited growth period is kept below 4, preferably in the range from about 2.0
to
about 3.5. The final stage is the production stage wherein either the "glucose
excess fed-batch mode" or the "mixed-feed fed-batch mode" can be used. In the
"glucose excess fed-batch mode," 2% glucose alone is added. In the "mixed-feed
fed-batch mode," a limiting amount of a non-inducing carbon source and glucose
is
added in the fermentor to induce the expression of the monellin gene driven by
a
GAP promoter.
The secreted monellin chimeric proteins can be recovered from the Pichia
pastoris culture medium by any methods known in the art. For example, methods
disclosed in U.S. Patent Nos. 3,878,184 and 3,998,798; Morris and Cagan,
Biochim. Biophys. Acta, 1972, 261:114-122; Kim et al., Protein Eng., 1989,
2:571-
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575; and Recently, Kondo et al., Nature Biotechnology, 1997, 15:453-457 can be
used for recovering and isolating the secreted monellin chimeric proteins. __
Preferably, the expressed and secreted chimeric protein is recovered by a
means
comprising ion-exchange chromatography. More preferably, the expressed and
5 secreted chimeric protein is recovered by a means comprising CM-Sephadex
column chromatography or DEAE-Sephadex column chromatography.
In another specific embodiment, the present invention provides the product
of the above processes.
10 6. EXAMPLE
6.1. Preparation of the Synthetic Recombinant Monellin DNA
The amino acid sequence of the recombinant monellin protein and the
nucleotide sequence of the DNA encoding the recombinant monellin protein are
shown in Figure 1. As shown in Figure 1, nucleotides I-150 encode residues 1-
50
15 of the B chain of the native monellin protein; nucleotides 150-152 encode
Glycine
as the linking "L" portion; and nucleotides 153-287 encode residues I-45 of
the A
chain of the native monellin protein. The recombinant monellin protein is
preceded
by the following amino acid sequence, which corresponds to a Met residue and
the
signal sequence of Saccharomyces cerevisiae mating pheromone a-factor:Met-Leu-
Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-
Glu-Lys-Arg-Glu-Ala-Glu-Ala-Glu-Phe (SEQ ID N0:3).
This synthetic DNA encoding the signal sequence of Saccharomyces
cerevisiae mating pheromone a-factor and the recombinant monellin protein was
prepared from the oligos MI-M4 and NI-N4, which were synthesized using the
Applied Biosystems 380B DNA Synthesizer by ACTG company (see Figures 2-3
and 5). The oligos were isolated by urea-polyacrylamide gel electrophoresis
and
purified by passing through a Sep-pak C18 column (Whatman) and annealed and
ligated as shown in Figure 3 to obtain the synthetic DNA bracketed by EcoRI
sites.
To synthesize the DNA encoding the signal sequence of Saccharomyces
cerevisiae mating pheromone a-factor and the recombinant monellin protein, in
100
ul PCR reaction volume, 2 pM of each of the oligo M2 to N3 were mixed with 10
pM M 1 and N4, heated to 94°C for 5 minutes in the absence of Tag DNA
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16
polymerase. The reaction mixture was then slowly cooled down to 37°C.
After 1
unit of the Vent DNA polymerase (New England Biolabs, Inc.) was added, the __
PCR reaction was performed according to the standard protocol. One hundred
microliter PCR reaction mixture contains 50 mM Tris-HCI (pH 8.0), 2.5 mM
MgCl.2, 10 mM DTT, 1 mM dNTP, and 1 unit of the Vent DNA polymerase. The
PCR reaction was performed as following: at 94°C for 1 minute,
53°C for 1.5
minutes, 72°C for 2 minutes within each cycle; and for a total of 30
cycles.
Finally, the reaction mixture was incubated at 72°C for 10 minutes. The
reaction
mixture was extracted by phenol/chloroform, precipitated with ethanol, and gel
purified in 1.2% low-melting agarose gel. The purified DNA fragment was
inserted into the pT7bleu (R) vector (Novagen, Inc.) to generate the pT7yM
plasmid (see Figure 5). In 20 ul DNA ligation reaction, 2 ul of 10 mM ATP, 40
units of the T4 DNA ligase (New England Biolab, Inc.) was added and mixed with
1 ug purified monellin DNA fragment and 50 ng pT7blue (R) vector. The reaction
mixture was kept at 16°C for 16 hours. The ligation mixture was
transformed into
host cells by adding 5 ul of the ligation mixture to 200 ul of E. coli
NovaBlue
competent cells (Messing, Methods in Enzymoloy, 1983, 101:20-78) and the
desired sequence was confirmed by dideoxy sequencing using T7 and U19 primer
(Sanger et al., Pnoc. Natl. Acad. Sci., 1985, 74:5463-5467).
(1)
6.2. Preparation of the Expression Vector pGWYS-1
The pGAPZa expression vector was purchased from Invitrogen, Inc. The
synthetic monellin DNA fragment was removed from pT7yMenallin with EcoRI
and inserted into an EcoRI site of the pGAPZa vector to give pGWYS. Briefly, 5
ug purified pT7yMenallion plasmid was digested in 20 ul reaction volume using
5
units EcoRI (Promega Inc.) at 37°C for 2 hours. After the reaction
mixture was
separated by 1 % low-melting agarose gel electrophoresis, the synthetic
monellin
DNA fragment was purified using the Wizard PCR Preps DNA purification kit
(Promega, Inc). One hundred ng purified monellin DNA fragment were used for
ligation into the expression pGAPZa vector. In the 10 ul ligation reaction, 50
ng
of the EcoRI digested pGAPZa vector was mixed with 100 ng purified monellin
DNA fragment. The ligation reaction was carried out in the presence of 10 ul
of
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20 mM Tris-HCl (pH 7.5), 10 mM MgCI, 10 mM DTT and 200 units of the T4
DNA ligase (New England Biolabs, Inc.) at 16°C overnight to give the
pGWYS.
The ligation mixture was transformed into E. coli TOP10F' cells (Invitrogen,
Inc.).
Twenty Zeocin-resistant clones were picked and orientation of the insert was
screened by PCR reaction using the a-factor ( 5'CTATTGCCAGCATTGCTGC3')
(SEQ ID N0:4) and the N4 oligos. Each selected clone was transferred into 3 ml
LB culture medium containing 200 ug/ml Zeocin and incubated with shaking at
37°C overnight. The recombinant plasmid pGWYS was prepared from 1.5 ml
cultured bacteria cells using the Qiagen Tip 20 kit system (Qiagen, Inc.).
Fifty ng
purified pGWYS plasmid was used as the PCR template to determine orientation
of
the insert. In 25 ul PCR reaction, 50 ng of the pGWYS plasmid was mixed with
2.5 pM of the a-factor and the N4 oligos in the presence of 1 unit Taq DNA
polymerase (Promega, Inc.). The PCR reaction was performed under the following
conditions: at 94°C for 1 minute, 55°C. for 1 minute,
72°C for 2 minutes within
each cycle; and for a total of 40 cycles. Finally, the reaction mixture was
incubated at 72°C. for 10 minutes. After the PCR reaction mixture was
separated
on 1.2% agarose gel, one of the clones which contains the insert with the
desired
orientation was named pGWYS-1. The sequence of the insert was further
confirmed by DNA sequencing.
6.3. Transformation of PicJria pastoris Cells with the pGWYS-1
To generate the high-level and stable expression of monellin in Pichia
pastoris, purified pGWYS-1 plasmid was transformed into Pichia pastoris cells
by
electroporation technique described in the Pichia pastoris Expression Kit
Manual
using the Electroporation Apparatus II (Invitrogen Inc.). Briefly, 500 ml of
the
Pichia pastoris GS115 cells were grown in YPD medium at 30°C to an
OD6~° of
1.3. Cells were pelleted with a centrifugation of 1,500 g for 5 minutes at
4°C.
Pelleted cells were washed with 500m1 of ice-cold sterile water. The washing
step
was repeated with 250 ml and 20 ml ice-cold sterile water, receptively. Then,
the
cells were washed with 20 ml of ice-cold 1 M sorbitol and resuspend in I ml
ice-
cold sorbitol. Forty ul of the yeast GS 115 cells in 1 M sorbitol were mixed
with
10 ug purified pGWYS-1 plasmid to total volume 50 ul and the mixture was
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transferred into an ice-cold cuvette. The cuvette containing the mixture was
incubated on ice for 5 minutes. Electroporation was performed according to the
Electroporation Apparatus II manual parameters (Invitrogen Inc manufacture).
After the electrical pulse, 1 ml of ice-cold 1 M sorbitol was added into the
cuvette,
and the content of the cuvette was transferred into a microcentrifuge tube.
Two
hundred ul transformed cells were plated on one 5 RDB plate containing 400
ug/ml
Zeocin. The plates were incubated at 30°C until colonies appeared.
Positive
transformants were characterized by their growth in the presence of Zeocin at
various concentrations, e.g., 400 ug/ml, 600 ug/ml, 800 ug/ml and 1000ug/ml.
6.4. Stability Test of the Positive Transformants
Three positive transformants were selected for further characterization based
on their growth in the presence of 800ug/ml Zeocin and the expression of
recombinant monellin by the 2% glucose induction. The following experiment was
performed to test their genetic stability. Each of these 3 positive
transformants was
picked up using a sterile toothpick and incubated on a YPD plate without any
selection at 30°C until colonies appeared. The colonies were picked up
and plated
on a new YPD plate until new colonies appeared. After such non-selective
growth
was repeated 50 times, each of the passage colonies was incubated on a
selective
plate containing 800 ug/ml Zeocin. The protein expression upon 2% glucose
induction was analyzed by SDS-PAGE. All three positive transformants showed
the same phenotype as the original colonies after 50 times passage on the YPD
plates.
6.5 Production of the Recombinant Monellin Protein
Each of the three positive transformants selected in 5.4. was grown inl liter
YPD medium in 5 liter flask at 30°C with vigorous shaking (250rpm).
Two ml
supernatant were obtained from the culture after 24 hours, 48 hours and 72
hours,
respectively. Five ul of the samples collected at each time point were
analyzed
using the 15-20% gradient polyacrylamide gel. The secreted recombinant
monellin
protein was observed as the 12 kD protein band. Quantitation of the SDS-PAGE
analysis using the Densitometer (Molecular Dynamic, Inc.) indicates that one
of the
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positive strain produced nearly 10 grams per liter secreted recombinant single-
chain
monellin protein. This strain was named GWyS 1. __
6.6 Purification of Secreted Recombinant Monellin Protein
Protein methods were used to purify the recombinant monellin protein
secreted from GwyS-1 yeast strain. According to the first method, after 72-
hour
culturing, supernatant was collected by a centrifugation at 12,000 rpm (
17,OOOg).
After collection, the supernatant pH was adjusted to about 6.8 using 0.1 N
NaOH
solution. One M NaH,PO,-Na,HPO, (pH6.8) was added into the supernatant till
1:100 (v/v) and mixed well. The supernatant was then loaded on the CM-Sephadex
column (Phamacia, Inc.) pre-equilibrated with 0.01 M NaH,PO,-Na,HPO, (pH6.8)
solution. After the column was washed with 5 column volume 0.01 M NaH,PO,-
Na,HPO, (pH6.8) solution, the recombinant monellin protein was eluted with 0.3
M
NaCI-0.01 M NaH,POa-Na.,HPO, (pH6.8) solution. After dialysis against water,
the
purity of the protein was determined to be about 98% by gel electrophoresis.
According to the second method, after 72-hour culturing, supernatant was
collected by a centrifugation at 12,000 rpm (17,OOOg). After collection, the
supernatant pH was adjusted to about 7.2 using 0.1 N NaOH solution. One M
NaCI-1 M NaH,PO~-Na,HPO~ (pH 7.2) was added into the supernatant till 1:100
(v/v) and mixed well. The supernatant was then loaded on the DEAE-Sephadex
column (Phamacia, Inc.) pre-equilibrated with 1 M NaH,PO,-Na,HPO, (pH 7.2)-1 M
NaCI solution. The flow-through fraction was collected and dialyzed against
water.
The purity of the protein was determined to be about 98% by gel
electrophoresis.
The recombinant moriellin protein purified according to either method was
further lyophilized to dry powder for testing its sweetness.
6.7. Sweetness and Stability Test
Sweetness of the purified recombinant monellin protein was assessed using
an ordinary taste test. Comparison to the sweetness of sucrose was made by
suitable dilutions on a weight basis. In a typical test, 1, 10, 25 and SO
mg/ml
aqueous sucrose solutions were used as standard solutions. The minimum weight
of
the purified recombinant monellin protein which could generate sweet taste was
compared with that of sucrose. The recombinant monellin of this invention
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requires the addition of an amount which is about 1000-fold less than that of
sucrose. For example, 50 ng/ml recombinant monellin protein solution was as --
sweet as 50 mg/ml sucrose (Lucky Supermarket's Lady Lee brand sugar).
Stability was measured by dissolving natural monellin (Sigma, Inc.) and the
5 purified recombinant monellin protein at 100 ug/ml concentration at pH 2.0,
4.0,
6.3. and 7.5. Each sample was heated to 37°C, SO°C, 60°C,
70°C, 80°C, 90°C and
100°C for 15 minutes and let cool to room temperature before tasting.
The most
dramatic difference was that natural monellin lost its sweetness when heated
to
50°C at pH 2.0, while the purified recombinant monellin protein
retained its
10 sweetness even after heating at 100°C for 5 minutes.
The present invention is not to be limited in scope by the microorganism
deposited or the specific embodiments described herein. Indeed, various
15 modifications of the invention in addition to those described herein will
become
apparent to those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall within the scope
of
the appended claims.
Various references are cited herein, the disclosures of which are
20 incorporated by reference in their entireties.
