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
1999-03-03
2001-02-06
Schwartman, Robert S. (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...
C435S069900
Reexamination Certificate
active
06183989
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for making desired polypeptides in yeast by expressing fusion polypeptides containing a monobasic protease processing site.
Background of the Invention
Yeast organisms produce a number of proteins synthesized intracellularly, but having a function outside the cell. These extracellular proteins are referred to as secreted proteins. Initially the secreted proteins are expressed inside the cell in the form of a precursor or a pre-protein containing a pre-peptide sequence ensuring effective direction of the expressed product (into the secretory pathway of the cell) across the membrane of the endoplasmic reticulum (ER). The pre-sequence, normally referred to as a signal peptide, is generally cleaved off from the desired product during translocation. Once having entered the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi apparatus the proteins are distributed to the plasma membrane, lysosomes and secretory vesicles.
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European published patent application No. 116 201 describes a process by which proteins heterologous to yeast are expressed, processed and secreted by a yeast host transformed by an expression vehicle harboring DNA encoding the desired protein, a leader sequence and a processing signal, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium, the leader sequence being a yeast &agr;-factor leader sequence.
The
Saccharomyces cerevisiae
MF&agr;1 (&agr;-factor) is synthesized as a pre-pro form of 165 amino acids comprising a signal or prepeptide of 19 amino acids followed by a “leader” or propeptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (LysArg((Asp/Glu)Ala)
2-3
&agr;-factor)
4
(Elements et al., Gene, 106, 1991, pp. 267-272). This publication describes certain modifications of the KEX2 site with the purpose of improving the KEX2 processing.
The signal-leader part of the pre-pro MF&agr;1 has been widely employed to obtain synthesis and secretion of heterologous proteins in
S. cerevisiae.
European published patent application No. 301 669 describes a process by which leader-sequences, in particular &agr;-factor leaders, are directing the secretion of expressed heterologous polypeptides in yeast. EP 324 274 discloses an improvement in the efficiency of expression and secretion of heterologous polypeptides by truncating the glycosylated a-factor leader sequence.
The secreted polypeptides are routed so as to be exposed to a proteolytic processing system which cleaves the peptide bond at the carboxy end of two consecutive basic amino acid residues. This enzymatic activity is in
S. cerevisiae
encoded by the KEX2 gene (Julius, D. A. et al., GENE, 37, 1884b, pp. 1075). Processing of the product by the KEX2 protease is necessary for the secretion of active
S. cerevisiae
mating factor &agr;1 (MF&agr;1 or &agr;-factor) whereas KEX2 is not involved in the secretion of
S. cerevisiae
mating factor a.
WO 90/10075 and WO 95/35384 describe modifications around the KEX2 site. Furthermore WO 95/34666, WO 92/11378 and WO 90/13653 describe the option of utilizing a secondary processing site comprised of the amino acids Ile-Glu-Gly-Arg named FX
a
, in situations where the usage of KEX2 is impractical.
In Applied Microbiological Biotechnology 35 (1991) 771-776, Seeboth et al., describes a mechanism by which a-leader bound polypeptides in yeast are processed by soluble KEX2 in vitro, wherein KEX2 was made soluble by modifying the KEX2 gene, and thereby presenting a method of increasing the site-specific processing in vitro. In GENE, 170 (1996) 107-112, Kjeldsen et al. describe that insertion of a spacer peptide following the dibasic KEX2 site, creating a N-terminal extension of the polypeptide precursor, greatly facilitates the KEX2 processing and subsequently improves the yield of the polypeptide precursor.
A recognized problem with the above described processes is that the level of secretion may be too low or the proteolytic processing may be incorrect or incomplete resulting in lower yields of the desired product.
The object of the present invention is to provide a yeast expressing process ensuring high yields of a desired polypeptide.
SUMMARY OF THE INVENTION
This invention relates to a process for making a desired polypeptide in yeast by expressing and secretion of a leader bound polypeptide which does not contain a KEX2 processing site. The leader bound polypeptide comprises the native a-factor leader peptide and the desired polypeptide linked by a monobasic processing site and possibly a spacer peptide.
More specifically, the present invention is related to a process of making a desired polypeptide in yeast by culturing in a suitable culture medium a yeast strain containing an expression vector capable of expressing a sequence with the following formula
SP-LP-X
n
-PS-*polypeptide*
wherein
SP is a signal peptide;
LP is the native &agr;-factor leader peptide or a leader peptide being at least 85% homologue to the native &agr;-factor leader peptide;
PS is a monobasic processing site Lys or Arg;
X is a spacer peptide containing n amino acids;
n is 0 or an integer from 1 to 10; and
*polypeptide* is the desired polypeptide;
with the proviso that the spacer peptide X is not Ile-Glu-Gly, Leu-Pro, Lys-Lys-Leu-Ile-Asp (SEQ ID NO: 4), Ile-Asp or Pro-Gly-Asp-Pro (SEQ ID NO: 5) and with the further proviso that the spacer peptide X does not contain a KEX2 cleavage site or together with PS or LP constitutes a KEX2 cleavage site and, when n=0, the C-terminal of the leader peptide is not Lys, Arg, Ile-Glu-Gly, Leu-Pro, Lys-Lys-Leu-Ile-Asp (SEQ ID NO: 6), Ile-Asp or Pro-Gly-Asp-Pro (SEQ ID NO. 7), whereupon the leader bound polypeptide is cleaved at the processing site PS either in vivo during passage through the cell membrane or in vitro after secretion into the culture medium whereupon the desired polypeptide is isolated.
The present invention also relates to DNA sequences encoding the above leader bound polypeptide, expression vectors containing such DNA sequences and yeast strains transformed with such vectors.
REFERENCES:
patent: 5591640 (1997-01-01), Loison et al.
patent: 0 324 274 (1989-07-01), None
patent: WO 90/10075 (1990-09-01), None
patent: WO 90/13653 (1990-11-01), None
patent: WO 92/11378 (1992-07-01), None
patent: WO 95/34666 (1995-12-01), None
patent: WO 95/35384 (1995-12-01), None
patent: WO 98/28429 (1998-07-01), None
Bird, P, Gething, M-J, Sambrook, J. (1987) “Translocation in Yeast & Mammalian Cells;” 105: 2905-2914, (Dec. 1987).
Latchinian-Sadek, L., Thomas, D. (1993) “Expression, Purification & Characterization of the Yeast KEXI Gene Product,” JBC 268: 534-40, Jan. 1993.
Baba et al., Biochem. and Biophy. Res. Comm., vol. 184, No. 1, pp. 50-59 (1992).
Kjeldsen et al., Gene , vol. 170, pp. 107-112 (1996).
Clements et al., Gene, vol. 106, pp. 267-272 (1991).
Chaudhuri et al., Eur. J. Biochem., vol. 210, pp. 811-822 (1992).
Sleep et al., Bio/Tech., vol. 8, pp. 42-46 (Jan. 1990).
Bourbonnais et al., Journal of Biological Chemistry, vol. 266, No. 20, pp. 13203-13209 (Jul. 15, 1991).
Seeboth et al., Appl. Microbiol. Biotechnol. vol. 35, pp. 771-776 (1991).
Julius et al., Cell., vol., 37, pp. 1075-1089, (Jul. 1984).
Brandt Jacob
Vad Knud
Davis Katharine F
Gregg, Esq. Valeta A.
Novo Nordisk A S
Schwartman Robert S.
Zelson, Esq. Steve T.
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