U.S. patent application number 11/113441 was filed with the patent office on 2005-09-22 for flavor active modified thaumatin and monellin and methods for their production and use.
This patent application is currently assigned to International Flavors & Fragrances Inc.. Invention is credited to Bolen, Paul L., Cihak, Paul L., Hawn, Regina D., Kossiakoff, Nicolas, Miller, Kevin P., Scharpf, Lewis G. JR..
Application Number | 20050209443 11/113441 |
Document ID | / |
Family ID | 22841030 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050209443 |
Kind Code |
A1 |
Bolen, Paul L. ; et
al. |
September 22, 2005 |
Flavor active modified thaumatin and monellin and methods for their
production and use
Abstract
A modified thaumatin protein, a modified monellin protein and
antibodies thereto are provided. Further, nucleic acid sequences
and vectors capable of expressing thaumatin or monellin are
provided.
Inventors: |
Bolen, Paul L.; (Middletown,
NJ) ; Cihak, Paul L.; (Leonardo, NJ) ;
Scharpf, Lewis G. JR.; (Fair Haven, NJ) ; Miller,
Kevin P.; (Middletown, NJ) ; Kossiakoff, Nicolas;
(Chambourcy, FR) ; Hawn, Regina D.; (Matawan,
NJ) |
Correspondence
Address: |
INTERNATIONAL FLAVORS & FRAGRANCES INC.
521 WEST 57TH ST
NEW YORK
NY
10019
US
|
Assignee: |
International Flavors &
Fragrances Inc.
|
Family ID: |
22841030 |
Appl. No.: |
11/113441 |
Filed: |
April 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11113441 |
Apr 22, 2005 |
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10175389 |
Jun 20, 2002 |
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6913906 |
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11113441 |
Apr 22, 2005 |
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09224514 |
May 6, 1999 |
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6420527 |
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Current U.S.
Class: |
530/388.26 |
Current CPC
Class: |
C07K 14/43 20130101;
C07K 16/20 20130101; C07K 16/40 20130101; C07K 16/16 20130101 |
Class at
Publication: |
530/388.26 |
International
Class: |
C07K 016/40 |
Claims
The claimed invention is:
1. An antibody to a modified thaumatin protein of SEQ ID NO: 2,
wherein amino acid residues 19 through 26 of the thaumatin protein
of SEQ ID NO: 2 are replaced by an amino acid sequence selected
from the group consisting of an amino acid sequence of a beefy
meaty peptide of SEQ ID NO: 6 or an amino acid sequence of amino
acid residues 4 through 11 of a monellin protein of SEQ ID NO:
4.
2. The antibody of claim 1 wherein the antibody is monoclonal.
3. An antibody to a modified monellin protein wherein amino acid
residues 4 through 11 of the monellin protein of SEQ ID NO: 4 are
replaced by an amino acid sequence selected from the group
consisting of an amino acid sequence that differs from the amino
acid sequence of the monellin protein of SEQ ID NO: 4 by at least
four amino acid residues, an amino acid sequence of a beefy meaty
peptide of SEQ ID NO: 6, or an amino acid sequence of amino acids
19 through 26 of a thaumatin protein of SEQ ID NO: 2.
4. The antibody of claim 3 wherein the antibody is monoclonal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of currently pending U.S.
patent application Ser. No. 10/175,389, filed on Jun. 18, 2002, a
divisional of U.S. application Ser. No. 09/224,514, now U.S. Pat.
No. 6,420,527, which is incorporated by reference herein.
[0002] Throughout this specification, various references are
identified by author name in parentheses. The citation to the
reference corresponding to the identified author can be found in
the section entitled References Cited preceding the claims. The
references in that section are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] Thaumatin and monellin are flavor active and flavor
enhancing proteins. Thaumatin, as shown by thaumatin II, has a wide
variety of uses as a flavor enhancing agent. The octapeptide known
as "delicious peptide" or "beefy meaty peptide" (BMP) is reported
to enhance flavor and produces an umami and sour taste, especially
in beef. Monellin is recognized as a potently sweet protein. Recent
work indicates that the 19 to 26 amino acid region of thaumatin is
a possible active site for sweetness determination. (Slootstra et
al. 1995). Substitution of this region with other amino acid
sequences, particularly those having some homology with this region
of thaumatin and believed to affect taste, may alter the properties
of thaumatin to provide new and useful function.
[0004] Recent work with monellin indicates that the lysine located
at position 4 in the A chain is a highly likely candidate for
involvement as a component of the receptor interaction site of
monellin. (Suami et al. 1996). Subsitution of this region with
other amino acid sequences, particularly those having some homology
with this region of monellin and believed to affect taste, may
alter the properties of monellin to provide new and useful
function.
SUMMARY OF THE INVENTION
[0005] The present invention provides new flavor active proteins.
Specifically, the new proteins are modified versions of the
thaumatin and monellin proteins. In thaumatin II amino acids in the
region of 19 to 26 (amino acids 19-26 of SEQ ID NO:2), which
appears to be a taste active region, are replaced with other amino
acid sequences. Most important is the replacement of this region
with the sequences for the octapeptide known as "delicious peptide"
or "beefy meaty peptide," i.e. LYS-GLY-ASP-GLU-GLU-SER-LEU-ALA (SEQ
ID NO:6), and the sequence that comprises the segment of protein
from the fourth to the eleventh amino acid in the A chain of
monellin, i.e. LYS-GLY-TYR-GLU-TYR-GLN-LEU-TYR (amino acids 4-11 of
SEQ ID NO:4; SEQ ID NO:11). When four (4) of the 207 amino acids in
the thaumatin b protein amino acid sequence were changed, a protein
is produced that has a dramatic savory effect when evaluated with a
complex salt enhancer such as L-arginine, ammonium chloride,
tartaric acid, monopotassium glutamate, or ribotide. In the
presence of the salt enhancer, the new protein with the BMP
sequence has a beefy, meaty, brothy impression and mouth feel.
[0006] This invention further provides a modified monellin protein,
either as A and B chains or as a single joined chain, wherein the
amino acids in the region of four to eleven of the A chain (or the
homologous section of single chain monellin), which appears to be
the taste active region, are replaced with other amino acid
sequences. Most important is the replacement of this region with
the sequences for the octapeptide known as "delicious peptide" or
"beefy meaty peptide," i.e. LYS-GLY-ASP-GLU-GLU-SER-LEU-ALA (SEQ.
ID. NO. 6), and the sequence that comprises the segment of protein
from the amino acid region of 19 to 26 in thaumatin II, i.e.
LYS-GLY-ASP-ALA-ALA-LEU-ASP-ALA (amino acids 19-26 of SEQ. ID. NO.
2).
[0007] This invention further provides the DNA sequences encoding
the modified thaumatin and monellin proteins and for derivatives of
such proteins and the DNA sequences of the derivatives. It also
teaches methods for producing these proteins using genetic
recombination techniques which can be used in a variety of
microorganisms. According to the present invention, these proteins
are expressable in yeast transformed by vectors comprising the
nucleic acid sequences encoding the proteins. The microorganisms
which have been transformed to express these proteins can be used
to cultivate cell lines capable of producing the proteins on a
large scale. Furthermore, this invention teaches the use of these
proteins as flavor additives and enhancers in food.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 depicts the flavor protein homologous regions of
thaumatin, BMP and monellin I.
[0009] FIG. 2 shows an NBRF score histogram for BMP.
[0010] FIG. 3 is an NBRF horizontal map for a BMP protein identity
matrix. The alphanumeric sequence in the left most column
corresponds to the NBRF reference identification number. A list of
the NBRF references is included in a subsection of the References
Cited section which precedes the claims and these references are
hereby incorporated by reference.
[0011] FIG. 4(A)-(E) depicts a BMP NBRF aligned sequence comparison
where the corresponding NBRF number appears adjacent to the peptide
compared to BMP.
[0012] FIGS. 5(A)-(F) show various properties of native
thaumatin.
[0013] FIGS. 6(A)-(F) show various properties of modified
thaumatin.
[0014] FIG. 7(A)-(B) Thaumatin Nucleic Acid (SEQ ID NO:1) and (SEQ
ID NO:2) Amino Acid Sequences
[0015] FIG. 8 Monellin A Chain Nucleic Acid (SEQ ID NO:3) and (SEQ
ID NO:4) Amino Acid Sequences
[0016] FIG. 9 BMP Nucleic Acid (SEQ ID NO:5) and Amino Acid (SEQ ID
NO: 6) Sequences
[0017] FIG. 10 Monellin Single Chain Nucleic Acid (SEQ ID NO:7) and
Amino Acid (SEQ ID NO:8) Sequences
[0018] FIGS. 11(A)-(B) A Modified Thaumatin Nucleic Acid (SEQ ID
NO:9) and Amino Acid (SEQ ID NO:10) Sequences
DETAILED DESCRIPTION OF THE INVENTION
[0019] Thaumatin is a naturally occuring sweet-tasting peptide.
Modified thaumatin is a thaumatin-like protein wherein more than
one of the amino acids numbered 19-26 of thaumatin have been
substituted, changed or modified. In particular, the amino acids
numbered 19-26 of thaumatin are changed or modified to comprise
amino acids having the flavorful or flavor enhancing amino acid
region of another protein, polypeptide or peptide. In one
embodiment/the change of as few as four amino acids in modified
thaumatin provides significantly altered taste properties from
thaumatin.
[0020] Monellin is a naturally occuring sweet-tasting peptide.
Modified thaumatin is a monellin-like protein wherein more than one
of the amino acids numbered 4-11 of monellin have been substituted,
changed or modified. In particular, the amino acids numbered 4-11
of monellin are changed or modified to comprise amino acids having
the flavorful or flavor enhancing amino acid region of another
protein, polypeptide or peptide. In one embodiment, the change of
as few as four amino acids in modified monellin provides
significantly altered taste properties from monellin.
[0021] The octapeptide known as "delicious peptide" or "beefy meaty
peptide" (BMP) is reported to enhance flavor and produces a umami
and sour taste, especially in beef. BMP is an octapeptide that can
be used in place of the active site of monellin or thaumatin to
change or modify the flavor and flavor enhancing properties of
these naturally sweet tasting proteins. Derivatives of modified
thaumatin and monellin can differ from modified thaumatin and
monellin in amino acid sequence or in ways that do not involve
sequence, or both. Derivatives in amino acid sequence are produced
when one or more amino acids in naturally occurring thaumatin or
monellin is substituted with a different natural amino acid, an
amino acid derivative or non-native amino acid. Particularly
preferred embodiments include naturally occurring thaumatin or
monellin, or biologically active fragments of naturally occurring
thaumatin or monellin, whose sequences differ from the wild type
sequence by one or more conservative amino acid substitutions,
which typically have minimal influence on the secondary structure
and hydrophobic nature of the protein or peptide. Derivatives may
also have sequences which differ by one or more non-conservative
amino acid substitutions, deletions or insertions which do not
abolish lo the thaumatin or monellin biological activity.
Conservative substitutions (substituents) typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. The non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0022] Other conservative substitutions can be taken from Table 1
and yet others are described by Dayhoff in the Atlas of Protein
Sequence and Structure (1988).
1TABLE 1 Conservative Amino Acid Replacements For Amino Acid Code
Replace with any of Alanine A D-Ala, Gly, beta-ALa, L-Cys, D-Cys
Arginine R D-Arg, Lys, homo-Arg, D-homo-Arg, Met, D-Met, lie,
D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin,
D- Gln Aspartic Acid D D-Asp, D-Asn, Asn, GlU/D-Glu, Gln, D- Gin
Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D- Thr Glutamine Q
D-Gln/Asn/D-Asn, GlU/D-Glu{circumflex over ( )}sp/D- Asp Glutamic
Acid E D-GlU/D-Asp/Asp, Asn, D-Asn, Gin, D-Gln Glycine G Ala,
D-Ala, Pro, D-Pro, Beta-Ala/ Acp Isoleucine 1 D-Ile, Val, D-Val,
Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met
Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo- Arg, Met, D-Met, He,
D-Ile, Orn, D- Orn Methionine M D-Met, S-Me-Cys, He, D-Ile, Leu,
D-Leu, Val, D-Val, Norleu Phenylalanine F D-Phe, Tyr, D-Thr,
L-Dopa, His, D-His, Trp, D-Trp, Trans 3, 4 or 5- phenylproline, cis
3, 4 or 5 phenylproline Proline P D-Pro, L-I-thioazolidine-4-
carboxylic acid, D- or L-l- oxazolidine-4-carboxylic acid Serine S
D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met (O), D-Met (0), Val,
D-Val Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met (O)
D-Met (0), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,
D-His Valine V D-Val, Leu, D-Leu, He, D-Ile, Met, D- Met
[0023] Other derivatives within the invention are those with
modifications which increase peptide stability. Such derivatives
may contain, for example, one or more non-peptide bonds (which
replace the peptide bonds) in the peptide sequence. Also included
are: derivatives that include residues other than naturally
occurring L-amino acids, such as D-amino acids or non-naturally
occurring or synthetic amino acids such as beta or gamma amino
acids and cyclic derivatives. Incorporation of D-instead of L-amino
acids into the polypeptide may increase its resistance to
proteases. See, e.g., U.S. Pat. No. 5,219,990, incorporated by
reference herein.
[0024] The polypeptides of this invention may also be modified by
various changes such as insertions, deletions and substitutions,
either conservative or nonconservative where such changes might
provide for certain advantages in their use.
[0025] In other embodiments, derivatives with amino acid
substitutions which are less conservative may also result in
desired derivatives, e.g., by causing changes in charge,
conformation and other biological properties. Such substitutions
would include for example, substitution of hydrophilic residue for
a hydrophobic residue, substitution of a cysteine or proline for
another residue, substitution of a residue having a small side
chain for a residue having a bulky side chain or substitution of a
residue having a net positive charge for a residue having a net
negative charge. When the result of a given substitution cannot be
predicted with certainty, the derivatives may be readily assayed
according to the methods disclosed herein to determine the presence
or absence of the desired characteristics.
[0026] Derivatives within the scope of the invention include
proteins and peptides with amino acid sequences having at least
eighty percent homology with thaumatin or monellin. More preferably
the sequence homology is at least ninety percent, or at least
ninety-five percent.
[0027] Just as it is possible to replace substituents of the
scaffold, it is also possible to substitute functional groups which
decorate the scaffold with groups characterized by similar
features. These substitutions will initially be conservative, i.e.,
the replacement group will have approximately the same size, shape,
hydrophobicity and charge as the original group. Non-sequence
modifications may include, for example, in vivo or in vitro
chemical derivatization of portions of naturally occurring
thaumatin or monellin, as well as changes in acetylation,
methylation, phosphorylation, carboxylation or glycosylation.
[0028] In a further embodiment the protein is modified by chemical
modifications in which activity is preserved. For example, the
proteins may be amidated, sulfated, singly or multiply halogenated,
alkylated, carboxylated, or phosphorylated. The protein may also be
singly or multiply acylated, such as with an acetyl group, with a
farnesyl moiety, or with a fatty acid, which may be saturated,
monounsaturated or polyunsaturated. The fatty acid may also be
singly or multiply fluorinated. The invention also includes
methionine analogs of the protein, for example the methionine
sulfone and methionine sulfoxide analogs. The invention also
includes salts of the proteins, such as ammonium salts, including
alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate,
hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate,
bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl
sulfonate and benzensulfonate salts.
[0029] Derivatives of thaumatin or monellin may also include
peptidomimetics of thaumatin or monellin. Such compounds are well
known to those of skill in the art and are produced through the
substitution of certain R groups or amino acids in the 25 protein
with non-physiological, non-natural replacements. Such
substitutions may increase the stability of such compound beyond
that of the naturally occurring compound.
[0030] It will be appreciated from the present disclosure that
modified thaumatin, modified monellin and their derivatives
according to the present invention can be used to alter, vary,
fortify modify, enhance or otherwise improve the taste of a wide
variety of materials which are ingested, consumed or otherwise
organoleptically sensed.
[0031] The terms "alter" and "modify" in their various forms will
be understood herein to mean the supplying or imparting of a flavor
character or note to an otherwise bland, relatively tasteless
substance, or augmenting an existing flavor characteristic where
the natural flavor is deficient in some regard or supplementing the
existing flavor impression to modify its organoleptic
character.
[0032] The term "enhance" is intended herein to mean the
intensification (by the use of the modified thaumatin or modified
monellin and derivatives of the present invention) of a flavor or
aroma note or nuance in a foodstuff or dairy product or cheese
without changing the quality of said note or nuance.
[0033] The term "flavoring composition" is taken to mean one which
contributes a part of the overall flavor impression by
supplementing or fortifying a natural or artificial flavor in a
material or one which supplies substantially all the flavor and/or
aroma character to a consumable article.
[0034] The term "foodstuff" as used herein includes both solid and
liquid ingestible materials for man or animals which materials
usually do, but need not, have nutritional value. Thus, foodstuffs
include meats, gravies, soups, convenience foods, malt, alcoholic,
milk and dairy products, seafoods, candies, vegetables, animal
foods, veterinary products and the like.
[0035] The modified proteins and derivatives of the present
invention can be combined with conventional flavoring agents or
adjuvants. Such co-ingredients or flavor adjuvants are well known
in the art for such and have been extensively described in the
literature. Requirements of such adjuvants are: (1) that they be
non-reactive with the carboxylic acid mixture of the present
invention; (2) that they be organoleptically compatible with the
mixture of the present invention such that the flavor of the
mixture is not adversely affected by the use of the adjuvant; and
(3) that they be ingestibly acceptable and thus non-toxic or
otherwise non-deleterious. Apart from these requirements,
conventional materials can be used and broadly include other flavor
materials, vehicles, stabilizers, thickeners, surface active
agents, conditioners, and flavor intensifiers. The following terms
are used in accordance with their meanings in the art. DNA is
deoxyribonucleic acid whether single- or double-stranded.
Complementary DNA (cDNA) is DNA which has a nucleic acid-sequence
obtained from reverse transcription of messenger ribonucleic acid
(mRNA). Recombinant genetic expression refers to the methods by
which a nucleic acid molecule encoding a polypeptide of interest is
used to transform a host cell so that the host cell will express
the polypeptide of interest, A plasmid or vector can be used to
introduce a nucleic acid molecule into a host cell. A plasmid or
vector can comprise, but need not, in addition to the gene or
nucleic acid sequence of interest, a gene that expresses a
selectable marker or phenotype and a gene that can control (induce
or inhibit) the expression of the gene of interest under certain
conditions.
Recombination Methods
[0036] Recombinant expression vectors containing a nucleic acid
sequence encoding modified thaumatin or modified monellin can be
prepared using well known methods. The expression vectors include a
modified thaumatin or modified monellin DNA sequence operably
linked to suitable transcriptional or translational regulatory
nucleotide sequences, such as those derived from a mammalian,
microbial, viral, or insect gene. Examples of regulatory sequences
include transcriptional promoters, operators, or enhancers, an mRNA
ribosomal binding site, and appropriate sequences which control
transcription and translation initiation and termination.
Nucleotide sequences are "operably linked" when the regulatory
sequence functionally relates to the modified thaumatin or modified
monellin DNA sequence. Thus, a promoter nucleotide sequence is
operably linked to a modified thaumatin or modified monellin DNA
sequence if the promoter nucleotide sequence controls the
transcription of the modified thaumatin or modified monellin DNA
sequence. The ability to replicate in the desired host cells,
usually conferred by an origin of replication, and a selection gene
by which transformants are identified, may additionally be
incorporated into the expression vector.
[0037] In addition, sequences encoding appropriate signal peptides
that are not naturally associated with modified thaumatin or
modified monellin can be incorporated into expression vectors. For
example, a DNA sequence for a signal peptide (secretory leader) may
be fused in-frame to the modified thaumatin or modified monellin
sequence so that modified thaumatin or modified monellin is
initially translated as a fusion protein comprising the signal
peptide. A signal peptide that is functional in the intended host
cells enhances extracellular secretion of the modified thaumatin or
modified monellin polypeptide. The signal peptide may be cleaved
from the modified thaumatin or modified monellin polypeptide upon
secretion of modified thaumatin or modified monellin from the
cell.
[0038] Suitable host cells for expression of modified thaumatin or
modified monellin polypeptides include prokaryotes, yeast or higher
eukaryotic cells. Appropriate cloning and expression vectors for
use with bacterial, fungal, yeast, and mammalian cellular hosts are
described, for example, in Pouwels et al. Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation
systems could also be employed to produce modified thaumatin or
modified monellin polypeptides using RNAs derived from DNA
constructs disclosed herein.
[0039] Prokaryotes include gram negative or gram positive
organisms, for example, E. coli or Bacilli. Suitable prokaryotic
host cells for transformation include, for example, E. coli,
Bacillus subtilis, Salmonella typhimurium, and various other
species within the genera Pseudomonas, Streptomyces, and
Staphylococcus.
[0040] In a prokaryotic host cell, such as E. coli, a modified
thaumatin or modified monellin polypeptide may include an
N-terminal methionine residue to facilitate expression of the
recombinant polypeptide in the prokaryotic host cell. The
N-terminal Met may be cleaved from the expressed recombinant
modified thaumatin or modified monellin polypeptide.
[0041] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. To construct an expression vector using pBR322,
an appropriate promoter and a modified thaumatin or modified
monellin DNA sequence are inserted into the pBR322 vector. Other
commercially available vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega
Biotec, Madison, Wis., USA).
[0042] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase).sub.f lactose promoter system (Chang et al., Nature
275:615, 1978; and Goeddel et al., Nature 281:544, 1979), 5
tryptopban (trp) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression
system employs a phage .lambda.P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lambda.P.sub.L promoter include plasmid pHUB2 (resident in E.
coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1
(ATCC 53082)).
[0043] Modified thaumatin or modified monellin polypeptides
alternatively may be expressed in yeast host cells, preferably from
the Saccharomyces genus (e.g., S. cerevisiae). Other genera of
yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast
vectors will often contain an origin of replication sequence from a
2 .mu. yeast plasmid, an autonomously replicating sequence (ARS), a
promoter region, sequences for polyadenylation, sequences for
transcription termination, and a selectable marker gene. Suitable
promoter sequences for yeast vectors include, among others,
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes
(Hess et al., j. Adv. Enzyme Reg. 7:149, 1968; and Holland et al.,
Biochem. 17:4900, 1978), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EPA-73,657 or in Fleer et. al.,
Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology,
8:135-139 (1990). Another alternative is the glucose-repressible
ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674,
1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors
replicable in both yeast and E. coli may be constructed by
inserting DNA sequences from pBR322 for selection and replication
in E. coli (Amp.sup.r gene and origin of replication) into the
above-described yeast vectors.
[0044] The yeast .alpha.-factor leader sequence may be employed to
direct secretion of a modified thaumatin or modified monellin
polypeptide. The .alpha.-factor leader sequence is often inserted
between the promoter sequence and the structural gene sequence.
See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc.
Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP
324,274. Other leader sequences suitable for facilitating secretion
of recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence may be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0045] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929, 1978.
[0046] The Hinnen et al. protocol selects for Trp.sup.+
transformants in a selective medium, wherein the selective medium
consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2%
glucose, 10. mu.g/ml adenine and 20 .mu.g/ml uracil. Yeast host
cells transformed by vectors containing ADH2 promoter sequence may
be grown for inducing expression in a "rich" medium. An example of
a rich medium is one consisting of 1% yeast extract, 2% peptone,
and 1% glucose supplemented with 80 .mu.g/ml adenine and 80
.mu.g/ml uracil. Derepression of the ADH2 promoter occurs when
glucose is exhausted from the medium.
[0047] Mammalian or insect host cell culture systems could also be
employed to express recombinant modified thaumatin or modified
monellin polypeptides. Baculovirus systems for production of
heterologous proteins in insect cells are reviewed by Luckow and
Summers, Bio/Technology 6:47 (1988). Established cell lines of
mammalian origin also may be employed. Examples of suitable
mammalian host cell lines include the COS-7 line of monkey kidney
cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells,
C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO)
cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the
CV-l/EBNA-1 cell line derived from the African green monkey kidney
cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J.
10: 2821, 1991).
[0048] Transcriptional and translational control sequences for
mammalian host cell expression vectors may be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from Polyoma virus, Adenovirus 2, Simian Virus 40
(SV40), and human cytomegalovirus. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites may be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained
from a viral genome as a fragment which may also contain a viral
origin of replication (Fiers et al., Nature 273:113, 1978). Smaller
or larger SV40 fragments may also be used, provided the
approximately 250 bp sequence extending from the Hind III site
toward the Bgl I site located in the SV40 viral origin of
replication site is included.
[0049] Exemplary expression vectors for use in mammalian host cells
can be constructed as disclosed by Okayama and Berg (Mol. Cell.
Biol. 3:280, 1983). A useful system for stable high level
expression of mammalian cDNAs in C127 murine mammary epithelial
cells can be constructed substantially as described by Cosman et
al. (Mol. Immunol. 23:935, 1986). A useful high expression vector,
PMLSV N1l/N4, described by Cosman et al., Nature 312:768, 1984 has
been deposited as ATCC 39890. Additional useful mammalian
expression vectors are described in EP-A-0367566, and in U.S.
patent application Ser. No. 07/701,415, filed May 16, 1991,
incorporated by reference herein. The vectors may be derived from
retroviruses. In place of the native signal sequence, a
heterologous signal sequence may be added, such as the signal
sequence for IL-7 described in U.S. Pat. No. 4,965,195; the signal
sequence for IL-2 receptor described in Cosman et al., Nature
312:768 (1984); the IL-4 signal peptide described in EP 367,566;
the type I IL-1 receptor signal peptide described in U.S. Pat. No.
4,968,607; and the type II IL-1 receptor signal peptide described
in EP 460,846.
[0050] An isolated and purified, modified thaumatin or modified
monellin protein according to the invention may be produced by
recombinant expression systems as described above or purified from
naturally occurring cells. Modified thaumatin or modified monellin
can be substantially purified, as indicated by a single protein
band upon analysis by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). One process for producing modified thaumatin or
modified monellin comprises culturing a host cell transformed with
an expression vector comprising a DNA sequence that encodes
modified thaumatin or modified monellin under conditions sufficient
to promote expression of modified thaumatin or modified monellin.
Modified thaumatin or modified monellin is then recovered from
culture medium or cell extracts, depending upon the expression
system employed. As is known to the skilled artisan, procedures for
purifying a recombinant protein will vary according to such factors
as the type of host cells employed and whether or not the
recombinant protein is secreted into the culture medium. For
example, when expression systems that secrete the recombinant
protein are employed, the culture medium first may be concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a purification matrix such as a gel filtration medium.
Alternatively, an anion exchange resin can be employed, for
example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dextran,
cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable
cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Sulfopropyl groups are
preferred. Finally, one or more reversed-phase high performance
liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC
media, (e.g., silica gel having pendant methyl or other aliphatic
groups) can be employed to further purify modified thaumatin or
modified monellin. Some or all of the foregoing purification steps,
in various combinations, are well known and can be employed to
provide an isolated and purified recombinant protein.
[0051] It is possible to utilize an affinity column comprising a
modified thaumatin or modified monellin-binding protein to
affinity-purify expressed modified thaumatin or modified monellin
polypeptides. Modified thaumatin or modified monellin polypeptides
can be removed from an affinity column using conventional
techniques, e.g., in a high salt elution buffer and then dialyzed
into a lower salt buffer for use or by changing pH or other
components depending on the affinity matrix utilized.
[0052] Recombinant protein produced in bacterial culture is usually
isolated by initial disruption of the host cells, centrifugation,
extraction from cell pellets if an insoluble polypeptide, or from
the supernatant fluid if a soluble polypeptide, followed by one or
more concentration, salting-out, ion exchange, affinity
purification or size exclusion chromatography steps. Finally,
RP-HPLC can be employed for final purification steps. Microbial
cells can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0053] Transformed yeast host cells can be employed to express
modified thaumatin or modified monellin as a secreted polypeptide
in order to simplify purification. Secreted recombinant polypeptide
from a yeast host cell fermentation can be purified by methods
analogous to those disclosed by Urdal et al. (J. Chromatog.
296:171, 1984). Urdal et al. describe two sequential,
reversed-phase HPLC steps for purification of recombinant human
IL-2 on a preparative HPLC column.
[0054] Antisense or sense oligonucleotides comprising a
single-stranded nucleic acid sequence (either RNA or DNA) capable
of binding to a target modified thaumatin or modified monellin mRNA
sequence (forming a duplex) or to the modified thaumatin or
modified monellin sequence in the double-stranded DNA helix
(forming a triple helix) can be made according to the invention.
Antisense or sense oligonucleotides, according to the present
invention.sub.f comprise a fragment of the coding region of
modified thaumatin or modified monellin cDNA. Such a fragment
generally comprises at least about 14 nucleotides, preferably from
about 14 to about 30 nucleotides. The ability to create an
antisense or a sense oligonucleotide, based upon a cDNA sequence
for a given protein is described in, for example, Stein and Cohen,
Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques
6:958, 1988.
[0055] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of complexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means. The
antisense oligonucleotides thus may be used to block expression of
modified thaumatin or modified monellin proteins.
[0056] Antisense or sense oligonucleotides further comprise
oligo-nucleotides having modified sugar-phosphodiester backbones
(or other sugar linkages, such as those described in WO91/06629)
and wherein such sugar linkages are resistant to endogenous
nucleases. Such oligonucleotides with resistant sugar linkages are
stable in vivo (i.e., capable of resisting enzymatic degradation)
but retain sequence specificity to be able to bind to target
nucleotide sequences. Other examples of sense or antisense
oligonucleotides include those oligonucleotides which are
covalently linked to organic moieties, such as those described in
WO 90/10448, and other moieties that increases affinity of the
oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine). Further still, intercalating agents, such as
ellipticine, and alkylating agents or metal complexes may be
attached to sense or antisense oligonucleotides to modify binding
specificities of the antisense or sense oliginucleotide for the
target nucleotide sequence.
[0057] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by insertion of the antisense or sense
oligonucleotide into a suitable retroviral vector, then contacting
the cell with the retrovirus vector containing the inserted
sequence, either in vivo or ex vivo. 5 Suitable retroviral vectors
include, but are not limited to, the murine retrovirus M-MuLV, N2
(a retrovirus derived from M-MuLV), or or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see PCT Application US
90/02656).
[0058] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0059] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0060] A preferred recombinant expression system is the Pichia
pastoris expression system. The yeast Pichia pastoris, a microbial
eukaryote, has been developed into an expression system. As a
yeast, Pichia pastoris is as easy to use as E. coli, while having
the advantages of eukaryotic expression (e.g. protein processing,
folding, and posttranslational modifications). While possessing
these advantages, it is faster, easier, and cheaper to use than
other eukaryotic expression systems, such as baculovirus or
mammalian tissue culture, and generally gives higher expression
levels. P. pastoris is similar to the baker's yeast, Saccharomyces
cerevisiae, including having the advantages of molecular and
genetic manipulations, but with the added advantages of 10- to
100-fold higher heterologous protein expression levels and the
protein processing characteristics of higher eukaryotes.
[0061] Pichia pastoris is completely amenable to the genetic,
biochemical, and molecular biological techniques that have been
developed over the past several decades for S. cerevisiae with
little or no modification. In particular, transformation by
complementation, gene disruption and gene replacement techniquest
developed for S. cerevisiae work equally well for Pichia
pastoris.
[0062] The genetic nomenclature adopted for Pichia pastoris mirrors
that used for S. cerevisiae (unlike that of Sc. pombe). For
example, the gene from S. cerevisiae that encodes the enzyme
histidinol dehydrogenase is called the HIS4 gene and likewise the
homologous gene from Pichia pastoris that encodes the same enzyme
is called the Pichia pastoris HIS4 gene, and so on. there is a very
high degree of cross-functionality between Pichia pastoris and S.
cerevisiae. For instance, many S. cerevisiae genes have been shown
to genetically complement the comparable mutants in Pichia
pastoris, and vice versa (e.g. the Pichia pastoris HIS4 gene
functionally complements S. cerevisiae his4 mutants and the S.
cerevisiae HIS4 gene functionally complements Pichia pastoris his4
mutants; other cross-complementing genes that have been identified
include LEU2, ARG4, TRP1, and URA3).
[0063] Pichia pastoris as a Methylotropic Yeast Pichia pastoris,
representing one of four different genera of methylotropic yeasts,
which also include Candida/Hansenula, and Torulopsis, is capable of
metabolizing methanol as a sole carbon source. The first step in
the metabolism of methanol is the oxidation of methanol to
formaldehyde by the enzyme alcoholoxidase. Expression of this
enzyme, coded for by the AOX1 gene, is tightly regulated and
induced by methanol to very high levels, typically >30% of the
total soluble protein in cells grown with methanol as the carbon
source. The AOX1 gene has been isolated and a plasmid-borne version
of the AOX1 promoter is used to drive expression of the gene of
interest for heterologous protein expression.
[0064] Expression of the AOX1 gene is controlled at the level of
transcription. IN methanol grown cells approximately 5% of the
polyA+ RNA is from the AOX1 gene. The regulation of the AOX1 gene
is similar to the regulation of the GAL1 gene (and others) of S.
cerevisiae in that control involves both a repression/derepression
mechanism. However, unlike the situation in S. cerevisiae,
derepression alone of the AOX1 gene (i.e. absence of a repressing
carbon source such as glucose) is not sufficient to generate even
minute levels of expression from the AOX1 gene. The inducer,
methanol, is necessary for expression.
[0065] Use for Heteroloqous Protein Expression
[0066] Pichia pastoris has been used successfully to express a wide
range of heterologous proteins. Heterologous expression in Pichia
pastoris can be either intracellular or secreted. Secretion
requires the presence of a signal sequence on the expressed protein
to target it to the secretory pathway. While several different
secretion signal sequences have been used successfully, including
the native secretion signal present on some heterologous proteins,
success has been variable. To improve the chances for success, two
different vectors with different secretion signals are included in
this kit: The vector, pHIL-Sl, carries a native Pichia pastoris
signal from the acid phosphatase gene, PHO1. The vector, pPIC9,
carries the secretion signal from the S. cerevisiae mating factor
pre-pro peptide.
[0067] Another advantage of expressing secreted proteins is that
Pichia pastoris secretes very low levels of native proteins, that,
combined with the very low amount of protein in the Pichia growth
media, means that the secreted heterologous protein comprises the
vast majority of the total protein in the media and serves as the
first step in purification of the protein.
[0068] Like S. cerevisiae, linear DNA can generate stable
transformants of Pichia pastoris via homologous recombination
between the transforming DNA and regions of homology within the
genome. Such integrants show extreme stability in the absence of
selective pressure even when present as multiple copies.
[0069] The expression vectors included int his kit carry the HIS4
gene for selection and are designed to be linearized with a
restriction enzyme such that HIS+recombinants can be generated by
integration at the his4 locus (a non-deletion, very low spontaneous
reversion mutation) or at the AOX1 locus. Integration events at the
AOX1 locus can result in the complete removal of the AOX1 coding
region (i.e. gene replacement) that in turn results in a
recombinant phenotype of His.sup.+ Mut.sup.- (Mut.sup.- refers to
the methanol utilization minus phenotype caused by the loss of
alcohol oxidase activity encoded by the AOX1 gene that results in a
no growth or slow growth phenotype on methanol media).
His+transformants can be readily and easily screened for the
Mut.sup.- phenotype, indicating integration at the AOX1 locus. The
His.sup.+ Mut.sup.- clones can be further screened for expression
of the heterologous protein of interest.
[0070] A number of independently isolated His.sup.+ Mut.sup.-
recombinants are routinely screened for expression of the
heterologous protein of interest because of the observation of
clonal variation (or difference in levels of expressing
heterologous protein seen among different transformants with the
same phenotype (His.sup.+ Mut.sup.- )). In some cases this clonal
variation can be explained by a difference in the number of copies
of the integrated plasmid (i.e. more copies=more expressed
protein), but it is not simply copy number that determines protein
expression level. There are several examples where one or more
copies of the integrants express at the same level (and that level
is high), as well as examples where an increase in the integrant
copy number causes a decrease in the protein expression level. The
best method at this time is to identify a successfully expressing
clone among several (e.g. 10-20) His.sup.+ Mut.sup.- transformants
empirically. Some examples of heterologous protein expression
include:
2TABLE 2 Expression Where Protein (g/L) Expressed Reference Human
serum albumin 4.0 S Barr, et al (HSA) (1992) fl-galactosidase
20,000 (U/mg I Tschopp, et al total protein) (1987a) Hepatitis B
surface 0.4 I Cregg, et al antigen (HBSAg) (1987) Tumor Necrosis
10.0 I Sreekrishna, et Factor (TNF) al (1988) Invertase 2.3 S
Tschopp, et al (1987b) Bovine Iysozyme c2 0.55 S Digan, et al
(1989) Tetanus toxin 12.0 I Clare, et al fragment C (1991a)
Pertusis antigen 3.0 I Romanus, et al P69 (1991) Streptokinase 0.08
i Hagenson, et al (active) (1989) Human EGF 0.5 s Cregg, et al
(1993) Mouse EGF 0.45 S Claire, et al (1991b) Aprotinin 0.8 S
Vedvick, et al (1991) Kunitz protease 1.0 S Wagner, et al inhibitor
(1992) (S = secreted; I = intracellular)
[0071] Specifically, the invention provides a modified thaumatin
protein having a modified amino acid sequence of amino acids 19 to
26 selected from an amino acid sequence differing from amino acids
19 to 26 of thaumatin (SEQ ID NO:2) by at least four amino acids;
an amino acid sequence of beefy meaty peptide (SEQ ID NO:6); and an
amino acid sequence of amino acids 4 to 11 of monellin (SEQ ID
NO:4), derivatives of these proteins and nucleic acid sequence
encoding their expression.
[0072] It also provides a modified monellin protein having a
modified amino acid sequence of amino acids 4 to 11 selected from
an amino acid sequence differing from amino acids 4 to 11 of
monellin (SEQ ID NO:4) by at least four amino acids; an amino acid
sequence of beefy meaty peptide (SEQ ID NO:6); and an amino acid
sequence of amino acids 19 to 26 of thaumatin (SEQ ID NO:2),
derivatives of these proteins and nucleic acids encoding their
expression.
[0073] This invention provides antibodies to modified thaumatin. In
a preferred embodiment the antibodies are monoclonal.
[0074] The invention provides vectors capable of expressing the
modified thaumatin or the modified monellin in a transformed host
cell. In a preferred embodiment/the vector is pPIC9. A preferred
host cell is Pichia pastoris. Methods for performing the
transformation are also provided as are methods of culturing the
host cells and recovering the expressed proteins.
[0075] This invention will be better understood from the Examples
which follow. However one skilled in the art will readily
appreciate that the specific methods and results discussed are
merely illustrative of the invention as described more fully in the
claims which follow thereafter.
EXAMPLES
Homology Search
[0076] An amino acid sequence homology search was conducted to
identify regions of similarity among taste active proteins.
Specifically, homology searches were conducted using the eight
residue sequences that show substantial relatedness in thaumatin,
monellin 1 and the synthetic octapeptide BMP or delicious peptide.
Gurmarin, miraculin.sub.f curculin and mabinlin (all sweet
proteins) sequences were searched with broad criteria allowing 6
out of 8 mismatches and 2 offsets. The results indicated only weak
homology, usually 2 of 8 and sometimes 3 of 8 mismatches. However,
"monellin and thaumatin are the only two proteins known to have a
very high specificity for the human sweet taste receptor." (Murzin,
A. G. 1993)
[0077] Efforts to identify the active sites for sweetness of
thaumatin and monellin have not been successful. Approaches
involved searches for related amino acid sequences, immunological
cross reactivity studies, comparisons of x-ray crystallographic
3-dimensional structures, modified protein studies and peptide
fragment evaluations. Studies in which the active site for
sweetness was sought by evaluating fragments of monellin and
thaumatin were largely unsuccessful. Sweetness was not detected in
monellin fragments and only in a very large fragment of thaumatin
(about half of the molecule equivalent to 12 kDa) suggesting that
tertiary structure is essential to produce sweetness. Similarly,
attempts to identify the active site by chemical modification of
certain amino acids of native monellin and thaumatin have not
yielded a clear picture (although certain amino acids appear to be
involved) due to conformational changes that likely occur.
Recognizing the importance of tertiary structure, Kohmura et al.
(1992) took the approach of conservatively modifying monellin to
determine whether any of the aspartic acid (Asp) residues play a
critical role in sweetness. Aspartic acid was replaced with either
asparagine of L-aminobutyric acid which they believed would
maintain the original tertiary structure. Only replacement of the
Asp at position 7 in the B chain resulted in the loss of sweetness
and suggests this is the most likely active site. This site is on
the surface of the molecule and is distant from the sequence (B
chain, amino acids 4 to 11) showing homology to thaumatin and
synthetic octapeptide BMP.
[0078] As of the homology search, a review of the literature did
not indicate that the region of interest in thaumatin (amino acids
19-26) were a region of particular interest with regard to
sweetness or taste properties. Antibodies to monellin and thaumatin
cross-react which indicates a high specificity for at least one
epitope. An epitope can be defined by as few as three amino acids.
While this region only has two amino acids in common for thaumatin
and monellin, the overall conformation may be one that defines an
antibody site. Also, the N-terminal region in thaumatin has been
shown to affect the expression of sweetness where thaumatin has
been recombinantly expressed in other organisms. It was observed
that when thaumatin is compared to other thaumatin like proteins,
the region of homology to BMP and monellin is the region of
greatest 5 dissimilarity. (Verrips, C. T. 1992, pp. 226-227).
[0079] Octapeptide Homology Comparison
[0080] A search for sequences similar to BMP and and a region
monellin I was conducted against other "chemosensory" proteins,
i.e. curculin, miraculin, gurmarin and thaumatin. While the exact
homology was not identified, a region in the early part of
thaumatin sequence overlapped with the region of interest and BMP
and had an equivalent degree of homology. See FIG. 1. Cagan (1984)
thought this homology was not significant, but noted that
antibodies to thaumatin cross reacts with monellin and predicted
cross reactivity of an antimonellin antibody with BMP. BMP does not
act as a sweetner but as a flavor enhancer. Thaumatin, in addition
to providing sweetness, has taste enhancement or modification
properties. Since homology does not occur among all sweet tasting
proteins, it seems possible that this region may play a role in
taste enhancement. On this basis, monellin could also have taste
enhancement properties. Thus, this homology could be a primary
determiner of taste enhancement in these proteins and other sweet
tasting proteins without this homology would not have significant
taste enhancement properties.
[0081] A search of the 1992 NBRF protein database for BMP related
sequences showed only one of the fifty highest scores was
significant and that was the original BMP sequence. Thus, this
sequence appears to be unique. Other proteins showed extensive
homologies and occurred in a wide variety of proteins. It is
possible that this homology confers taste enhancement to these
proteins.
[0082] Modification of Thaumatin
[0083] A region was identified in thaumatin (residues 19-26) and
monellin (residues 4-11 in the A chain) that have 50% homology with
each other and with the octapeptide variously known as "delicious
peptide" or "beefy meaty peptide" (BMP). See SEQ ID NO:6. Modified
thaumatin was produced wherein the region of amino acids 19-26 of
thaumatin was modified to specify the sequence for the BMP
octapeptide to determine whether this modification changes the
taste qualities associated with thaumatin. A gene encoding the
modified thaumatin was used to transform yeast and was expressed as
a polypeptide to be tested for its effect on flavor perception.
[0084] This modified thaumatin was shown to yield a recombinant
protein that is not sweet (500 ppm) but alters the salty and savory
impression (10 ppm) of succinic acid and the impression of a salt
enhancer (IFF #30) comprising L-arginine, ammonium chloride,
tartaric acid, monopotassium glutamate and ribotide.
3TABLE 3 Salt Enhancer IFF #30. IPC Ingredient % as consumed Weight
14886 L-arginine Nat. "FLG" 0.17 320.75 14279 Ammnonium Chloride
0.2 377.36 200810 Tartaric acid Nat PWD "FLG" 0.08 37.74
Monopotassium glutamate 0.13 245.28 183920 Ribotide 0.01 18.87
Total 0.59 1000.00 Suggested Usage: 0.02% as consumed.
[0085]
4TABLE 4 Amino Acid Composition of Modified Thaumatin No. Percent
Non-polar: A 14 6.76 V 10 4.83 L 9 4.35 I 8 3.86 P 12 5.80 M 1 0.48
F 11 5.31 W 3 1.45 Polar: G 24 11.59 S 15 7.25 T 20 9.66 C 16 7.73
Y 8 3.86 N 8 3.86 Q 4 1.93 Acidic: D 8 6.28 E 13 3.86 Basic: K 0
5.31 R 12 5.80 H 11 0.00
[0086] The calculated molecular weight of modified thaumatin is
22,292,
[0087] For comparison, native thaumatin has the following amino
acid composition:
5TABLE 5 Amino Acid Composition of Thaumatin No. Percent Non-polar:
A 16 7.73 V 10 4.83 L 9 4.35 I 8 3.86 P 12 5.80 M 1 0.48 F 11 5.31
W 3 1.45 Polar: G 24 11.59 S 14 6.76 T 20 9.66 C 16 7.73 Y 8 3.86 N
8 3.86 Q 4 1.93 Acidic: D 14 6.76 E 6 2.90 Basic: K 11 5.31 R 12
5.80 H 0 0.00
[0088] Thaumatin has a calculated molecular weight of 22204.
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[0090] 2. de Vos, A. M. et al. (1985) "Three Dimensional Structure
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[0091] 3. Iijima, H. (1995) "Design and Protein Engineering of a
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[0092] 4. Iyengar, R. B. (1979) "The Complete Amino Acid Sequence
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[0095] 7. Kuramitsu, R. et al. (1993) "New Usage of Aspartic Acid
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[0106] 3. Alestrom P. et al. (1981), Hexon protein--Human
adenovirus 2, Atlas November 1982, HXAD2NBRF;
[0107] 4. Alin P. et al. (1989), Gluthathione transferase 8,
cytosolic--Rat, Biochem. J., 261:531-439, XURT8CNBRF;
[0108] 5. Allison L. A. et al. (1985), DNA-directed RNA polymerase
II 215K polypeptide--Yeast (Saccharomyces cerevisiae), Cell
42:599-610, RNBY2LNBRF;
[0109] 6. Aoki I. et al. (1991), Eosinophil granule major basic
protein 1 precursor--Guinea pig, FEBS Lett., 279:330-334,
S13625NBRF;
[0110] 7. Aoki I. et al. (1991), Eosinophil granule major basic
protein 2 precursor--Guinea pig, FEBS Lett., 282:56-60,
S15102NBRF;
[0111] 8. Au-Young J. et al. (1990), Chitin synthase--Imperfect
fungus (Candida albicans), Mol. Microbiol., 4:197-207,
S11808NBRF;
[0112] 9. Baer R. et al. (1984), BRRF2 protein--Epstein-Barr virus
(strain B95-8), Nature, 310:207-211, QQBE30NBRF;
[0113] 10. Bankier A. T. et al. (1983), Thymidine
kinase--Epstein-Barr virus (strain B95-8), EMBO J., 5:1959-1966,
KIBETENBRF;
[0114] 11. Begg G. S., et al. (1978), Connective-tissue activating
peptide III--Human, Proc. Natl. Acad. Sci U.S.A. Biochemistry,
80:765-769, TGHUNBRF;
[0115] 12. Benfield P. A. et al. (1984), Creatine kinase M
chain--Rat, J. Biol. Chem., 259:14979-14984, KIRTCMNBRF;
[0116] 13. Benfield P. A. et al. (1988), Creatine kinase (CK)--Rat,
Gene, 63:227-243, JT0277NBRF;
[0117] 14. Buskin J. N., et al. (1985), Creatine kinase M
chain--Mouse, J. Mol. Evol., 22:334-341, A2359ONBRF;
[0118] 15. Calabrese L., et al. (1989), Superoxide dismutase
(Cu-Zn)--Blue shark, FEBS Lett, 50:49-52, S04623NBRF;
[0119] 16. Citron B. A. et al. (1984), Galactokinase--Yeast
(Saccharomyces cerevisiae), J. Bacteriol., 158:269-278,
KIBYGGNBRF;
[0120] 17. Dawson P. A. et al. (1989), Oxysterol-binding
protein--Rabbit, J. Biol. Chem., 264:16798-16803, A34404NBRF;
[0121] 18. Fang J. K., et al. (1980), 3-Hydroxyacyl--CoA
dehydrogenase--Pig, submitted to the Atlas, October 1982,
116-196-198, DEPGCNBRF;
[0122] 19. Ghosh S. et al. (1990), Transcription factor
NF-kappaB--Mouse, Cell, 62:1019-1029, A35697NBRF;
[0123] 20. Gitt M. A., et al. (1985), DNA-directed RNA polymerase
sigma chain--Bacillus subtilits, J. Biol. Chem, 20:7178-7185,
A22626NBRF;
[0124] 21. Gustafson G. et al. (1989), Alpha-a protein--Barley
stripe mosaic virus, Virology, 170:370-377, PAVBBSNBRF;
[0125] 22. Haas R. C. et al. (1990), Creatine kinase precursor,
sarcomere-specific, mitochondrial--Human, J. Biol. Chem.
265:6921-6927, A35756NBRF;
[0126] 23. HeIfman O. M. et al. (1985), Tropomyosin 1, smooth
muscle--Chicken, J. Biol. Chem., 259:14136-14143, TMCHS1NBRF;
[0127] 24. Herring B. P. et al. (1990), Myosin-light-chain kinase,
skeletal muscle--Rabbit, J. Biol. Chem., 265:1724-1730,
A35021NBRF;
[0128] 25. Hirsch-Behnam A. et al. (1990), Hypothetical protein
El--Human papillomavirus, Virus Res., 18:81-98, S15616NBRF;
[0129] 26. Holt J. C., et al. (1986), Platelet basic
protein--Human, Biochemistry, 25:1988-1996, A24448NBRF;
[0130] 27. Hossle J. P., et al. (1986), B-creatine kinase
protein--Chicken, Nucleic Acids Res., 14:1449-1463, A24793NBRF;
[0131] 28. Hossle J. P., et al. (1988), Creatine kinase precursor,
mitochondrial--Chicken (fragment), Biochem. Biophys. Res. Commun.
151:408-416; A27708NBRF;
[0132] 29. Jofuku K. D. et al. (1989), Trypsin inhibitor KTi3+
(Kunitz)--Soybean, Plant Cell, 1:427-435, JQ0968NBRF;
[0133] 30. Jornvall H. et al. (1981), Hexon protein--Human
adenovirus 2, J. Biol. Chem., 256:6181-6186, HXAD2NBRF;
[0134] 31. Klein A., et al. (1988), Methyl coenzyme M reductase
beta chain--Methanococcus voltai, submitted to the EMBL Data
Library, S03257NBRF;
[0135] 32. Larimer F. W. et al. (1989), revl protein--Yeast
(Saccharomyces cerevisiae), J. Bacteriol., 171-230-237,
A3224ONBRF;
[0136] 33. Lehman L. J. et al. (1990), Dinitrogenase
reductase--Rhodospirillum rubrum, Gene, 95:143-147, JW0039NBRF;
[0137] 34. Levin D. E., et al. (1990),. Protein kinase 1--Yeast,
Proc. Natl. Acad. Sci. U.S.A., 87:8272-8276, A36474NBRF;
[0138] 35. Lin C. S., et al. (1988), L-plastin--Human, Mol. Cell.
Biol. 8:4659-4668, A31559NBRF;
[0139] 36. Mariman E. C. M. et al. (1989), Creatine kinase chain
B--Human, Nucleic Acids Res., 17-6385, S15935NBRF;
[0140] 37. Mariman E. C. M., et al. (1987), Creatine kinase B
chain--Human, Genomics, 1:126-137, A27174NBRF;
[0141] 38. Michaels M. L. et al. (1990), Adenine
glycosylase--Escherichia coli, Nucleic Acids Res., 18:3841-3845,
JQ0546BNRF;
[0142] 39. Mukai H. et al. (1991), Calcineurin B-like protein--Rat,
Biochem. Biophys. Res. Commun., 179:1325-1330, JQ1232NBRF;
[0143] 40. Nambu J. R., et al. (1986), Egg-laying hormone -1
precursor--Sea hare, J. Neurosci, 6:2026-2036, A26147NBRF;
[0144] 41. Nishiya Y., et al. (1990), Neutral proteinase--Bacillus
stearothermophilus, J. Bacteriol. 172:4861-4869, B36706NBRF;
[0145] 42. Payne R. M. et al. (1991), Creatine kinase--Rat,
Biochem. Biophys. Acta, 1089:352-361, S17188NBRF;
[0146] 43. Pentecost B. T. et al. (1990), Creatine kinase B
chain--Rat, Mol. Endocrinol., 4:1000-1010, A35682NBRF;
[0147] 44. Perryman M. B., et al. (1986), Creatine kinase M
chain--Human, Biochem. Biophys. Res. Commun., 140:981-989,
A26387NBRF;
[0148] 45. Pickering L. et al. (1985), Creatine kinase B
chain--Rabbit, Proc. Natl. Acad. Sci. U.S.A., 82:2310-2314,
KIRBCBNBRF;
[0149] 46. Roman D., et al. (1985), Creatine Kinase M chain--Dog,
Proc. Natl. Acad. Sci U.S.A., 82:8394-8398, A24685NBRF;
[0150] 47. Rupp F. et al. (1991), Agrin--Rat, Neuron, 6:811-823,
JH0399BNRF;
[0151] 48. Sanders C. et al. (1985), Tropomyosin 1, smooth
muscle--Chicken, J. Biol. Chem., 260:7264-7275, TMCHS1NBRF; 49.
Takio K., et al. (1986), Myosin light chain kinase, skeletal
muscle--Rabbit, Biochemistry , 25:8049-8057;
[0152] 50. Tamura M., et al. (1989), Delicious peptide--Bovine,
Agric. Biol. Chem., 53:319-325, JT0438NBRF;
[0153] 51. Trask R. V., et al. (1988), Creatine kinase M
chain--Human, J. Biol. Chem., 263:17142-17149NBRF;
[0154] 52. Tsai-Wu J. J. et al. (1991), MicA protein--Escherichia
coli, J. Bacteriol., 173:1902-1910, B38535NBRF; and 53. Zakin M.
M., et al (1983), metL bifunctional enzyme--Escherichia Coli,
258:3028-3031, DEECK2NBRF.
Sequence CWU 1
1
12 1 621 DNA Bos sp. 1 gctaccttcg aaatcgttaa cagatgttct tacactgttt
gggctgctgc ttccaagggt 60 gacgctgctt tggacgccgg tggtagacaa
ttgaactctg gtgaatcctg gaccatcaac 120 gtcgaaccag gtaccaaggg
tggtaagatc tgggctagaa ccgactgtta cttcgatgac 180 tctggttccg
gtatctgtaa gactggtgac tgtggtggtt tgttgagatg taagagattc 240
ggtagaccac caaccacttt ggctgaattc tctttgaacc aatacggtaa ggactacatc
300 gatatctcca acatcaaggg tttcaacgtt ccaatggact tctctccaac
cactagaggt 360 tgtagaggcg tcagatgtgc tgctgacatc gttggtcaat
gtccagctga ccttaaggct 420 ccaggtggtg gttgtaacga cgcttgtacc
gttttccaaa cttccgaata ctgttgtacc 480 actggtaagt gtggtccaac
cgaatactct agattcttca agagattgtg tccagacgct 540 ttctcctacg
tcttggacaa gccaactacc gtcacttgtc caggttcttc caactacaga 600
gttaccttct gtccaactgc c 621 2 207 PRT Bos sp. 2 Ala Thr Phe Glu Ile
Val Asn Arg Cys Ser Tyr Thr Val Trp Ala Ala 1 5 10 15 Ala Ser Lys
Gly Asp Ala Ala Leu Asp Ala Gly Gly Arg Gln Leu Asn 20 25 30 Ser
Gly Glu Ser Trp Thr Ile Asn Val Glu Pro Gly Thr Lys Gly Gly 35 40
45 Lys Ile Trp Ala Arg Thr Asp Cys Tyr Phe Asp Asp Ser Gly Ser Gly
50 55 60 Ile Cys Lys Thr Gly Asp Cys Gly Gly Leu Leu Arg Cys Lys
Arg Phe 65 70 75 80 Gly Arg Pro Pro Thr Thr Leu Ala Glu Phe Ser Leu
Asn Gln Tyr Gly 85 90 95 Lys Asp Tyr Ile Asp Ile Ser Asn Ile Lys
Gly Phe Asn Val Pro Met 100 105 110 Asp Phe Ser Pro Thr Thr Arg Gly
Cys Arg Gly Val Arg Cys Ala Ala 115 120 125 Asp Ile Val Gly Gln Cys
Pro Ala Asp Leu Lys Ala Pro Gly Gly Gly 130 135 140 Cys Asn Asp Ala
Cys Thr Val Phe Gln Thr Ser Glu Tyr Cys Cys Thr 145 150 155 160 Thr
Gly Lys Cys Gly Pro Thr Glu Tyr Ser Arg Phe Phe Lys Arg Leu 165 170
175 Cys Pro Asp Ala Phe Ser Tyr Val Leu Asp Lys Pro Thr Thr Val Thr
180 185 190 Cys Pro Gly Ser Ser Asn Tyr Arg Val Thr Phe Cys Pro Thr
Ala 195 200 205 3 135 DNA Bos sp. 3 ttcagagaaa ttaaggggta
cgaataccaa ttgtatgttt acgcttctga caagcttttc 60 agagctgaca
tttctgaaga ctacaagacc cgcggtagaa agttgttgag attcaacggt 120
ccagttccac cacca 135 4 45 PRT Bos sp. 4 Phe Arg Glu Ile Lys Gly Tyr
Glu Tyr Gln Leu Tyr Val Tyr Ala Ser 1 5 10 15 Asp Lys Leu Phe Arg
Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 20 25 30 Arg Lys Leu
Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 35 40 45 5 24 DNA Bos sp. 5
aagggtgacg aagaatcttt ggct 24 6 8 PRT Bos sp. 6 Lys Gly Asp Glu Glu
Ser Leu Ala 1 5 7 285 DNA Bos sp. 7 atgggagaat gggaaattat
cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaag
aaaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120
ccatgtatga agaagactat ttacgaaaac gaaagagaaa ttaaggggta cgaataccaa
180 ttgtatgttt acgcttctga caagcttttc agagctgaca tttctgaaga
ctacaagacc 240 cgcggtagaa agttgttgag attcaacggt ccagttccac cacca
285 8 95 PRT Bos sp. 8 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro
Phe Thr Gln Asn Leu 1 5 10 15 Gly Lys Phe Ala Val Asp Glu Glu Asn
Lys Ile Gly Gln Tyr Gly Arg 20 25 30 Leu Thr Phe Asn Lys Val Ile
Arg Pro Cys Met Lys Lys Thr Ile Tyr 35 40 45 Glu Asn Glu Arg Glu
Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Tyr 50 55 60 Ala Ser Asp
Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr 65 70 75 80 Arg
Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 9
621 DNA Artificial Sequence Description of Artificial Sequence
Synthetic 9 gctaccttcg aaatcgttaa cagatgttct tacactgttt gggctgctgc
ttccaagggt 60 gacgaggagt ctttggccgg tggtagacaa ttgaactctg
gtgaatcctg gaccatcaac 120 gtcgaaccag gtaccaaggg tggtaagatc
tgggctagaa ccgactgtta cttcgatgac 180 tctggttccg gtatctgtaa
gactggtgac tgtggtggtt tgttgagatg taagagattc 240 cgtagaccac
caaccacttt ggctgaattc tctttgaacc aatacggtaa ggactacatc 300
gatatctcca acatcaaggg tttcaacgtt ccaatggact tctctccaac cactagaggt
360 tgtagaggcg tcagatgtgc tgctgacatc gttggtcaat gtccagctga
ccttaaggct 420 ccaggtggtg gttgtaacga cgcttgtacc gttttccaaa
cttccgaata ctgttgtacc 480 actggtaagt gtggtccaac cgaatactct
agattcttca agagattgtg tccagacgct 540 ttctcctacg tcttggacaa
gccaactacc gtcacttgtc caggttcttc caactacaga 600 gttaccttct
gtccaactgc c 621 10 207 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 10 Ala Thr Phe Glu Ile Val Asn Arg
Cys Ser Tyr Thr Val Trp Ala Ala 1 5 10 15 Ala Ser Lys Gly Asp Glu
Glu Ser Leu Ala Gly Gly Arg Gln Leu Asn 20 25 30 Ser Gly Glu Ser
Trp Thr Ile Asn Val Glu Pro Gly Thr Lys Gly Gly 35 40 45 Lys Ile
Trp Ala Arg Thr Asp Cys Tyr Phe Asp Asp Ser Gly Ser Gly 50 55 60
Ile Cys Lys Thr Gly Asp Cys Gly Gly Leu Leu Arg Cys Lys Arg Phe 65
70 75 80 Gly Arg Pro Pro Thr Thr Leu Ala Glu Phe Ser Leu Asn Gln
Tyr Gly 85 90 95 Lys Asp Tyr Ile Asp Ile Ser Asn Ile Lys Gly Phe
Asn Val Pro Met 100 105 110 Asp Phe Ser Pro Thr Thr Arg Gly Cys Arg
Gly Val Arg Cys Ala Ala 115 120 125 Asp Ile Val Gly Gln Cys Pro Ala
Asp Leu Lys Ala Pro Gly Gly Gly 130 135 140 Gly Asn Asp Ala Cys Thr
Val Phe Gln Thr Ser Glu Tyr Cys Cys Thr 145 150 155 160 Thr Gly Lys
Cys Gly Pro Thr Glu Tyr Ser Arg Phe Phe Lys Arg Leu 165 170 175 Cys
Pro Asp Ala Phe Ser Tyr Val Leu Asp Lys Pro Thr Thr Val Thr 180 185
190 Cys Pro Gly Ser Ser Asn Tyr Arg Val Thr Phe Cys Pro Thr Ala 195
200 205 11 8 PRT Bos sp. 11 Lys Gly Tyr Glu Tyr Gln Leu Tyr 1 5 12
8 PRT Bos sp. 12 Lys Gly Asp Ala Ala Leu Asp Ala 1 5
* * * * *