Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-26
2004-08-24
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C435S254100, C536S023400, C536S023100, C530S350000
Reexamination Certificate
active
06780615
ABSTRACT:
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
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.
2. BACKGROUND ART
2.1. Monellin
Monellin belongs to a family of intensely sweet proteins derived from tropical plants (Dansby,
Nature 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. Pat. Nos. 3,878,184 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).
U.S. Pat. No. 4,300,576 discloses smoking articles containing thaumatin or monellin. U.S. Pat. No. 4,562,076 discloses chewing gum with coating of thaumatin or monellin. U.S. Pat. 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,
Nature 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. Pat. No. 5,234,834 discloses constructs for expression of single-chain monellin in plant cells. U.S. Pat. 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 1-46 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. Pat. No. 5,487,983 discloses an expression system for making the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. No. 5,670,339 discloses DNA encoding the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. No. 5,672,372 discloses methods for sweetening a food composition with the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. 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.
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. Pat. No. 4,837,148 discloses autonomous replication sequences for
Pichia pastoris
. U.S. Pat. No. 4,855,231 discloses regulatory region for heterologous gene expression in
Pichia pastoris
cells. U.S. Pat. No. 4,882,279 discloses site selective genomic modification of
Pichia pastoris
. U.S. Pat. No. 4,929,555 discloses a method for making whole cells of
Pichia pastoris
competent for transformation. U.S. Pat. No. 5,122,465 discloses a process for generating a selectable phenotype in strains of
Pichia pastoris
. U.S. Pat. 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
Aspergillus giganteus
alpha-Sarcin (Martinez-Ruiz et al.,
Protein Expr. Purif
., 1998, 12(3):315-22; Abdulaev et al.,
Protein Expr. Purif
., 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. Biophys. Acta
., 1998, 1396(3):306-19), the signal sequence of the mouse alpha-factor signal (cCell) 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, 9(2):159-70), signal sequence of mouse major urinary protein (Ferrari et al.,
FEBS Lett
., 1997, 401(1):73-7), signal sequence of pho1 (Skory et al.,
Curr. Genet
., 1996, 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 &agr;-factor (Fidler et al.,
J. Mol. Endocrinol
., 1998, 21(3):327-336).
Although the methylotrophic yeast
Pichia pastoris
has been used successfully for the production of various heterologous proteins, U.S. Pat. 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. Pat. No. 5,324,639 further discloses that it is especially difficult to foresee if a particular protein will be secreted by the methylotrophic yeast host, and if it i
Genway Biotech Inc.
Katcheves Konstantina
Ketter James
Morrison & Foerster / LLP
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