Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1998-02-24
2000-08-22
Brusca, John S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 9141, 43525411, 4352555, 4352542, 43525421, 43525423, 4352523, 435348, 435349, 435325, 536 241, C12P 2100, C12N 510, C12N 111, C12N 1531
Patent
active
061070572
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Recombinant DNA technology has revolutionized the ability to produce polypeptides economically. Yeast host cells and expression systems are useful for such production. Examples of yeast expression systems are Brake, U.S. Pat. No. 4,870,008; Cregg, U.S. Pat. No. 4,837,148; Stroman et al., U.S. Pat. No. 4,855,231; Stroman et al. U.S. Pat. No. 4,879,231; Brierley et al., U.S. Pat. No. 5,324,639; Prevatt et al., U.S. Pat. No. 5,330,901; Tschopp, EP 256 421; Sreekrishna et al., J. Basic Microbiol. 28(1988): 4 265-278; Tschopp et al., Bio/Technology 5(1987): 1305-1308; Cregg et al., Bio/Technology 5(1987): 479-485; Sreekrishna et al. Biochemistry 28(1989): 4117-4125; and Bolen et al., Yeast 10: 403-414 (1994).
General recombinant DNA methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a polynucleotide molecule comprising a first nucleotide sequence that encodes at least a fragment of a leader sequence and a second nucleotide sequence that encodes a polypeptide heterologous to the leader sequence, comprises an amino acid sequence that comprises at least about 70% sequence identity to the leader sequence of Pichia acaciae killer toxin, leader sequence, and suitable for expression thereof, the heterologous polypeptide is produced that is free of additional N-terminal amino acids.
The polynucleotide of the invention can be used to construct expression vectors and host cells capable of producing the polynucleotide or expressing the desired polypeptide.
Yet another object of the invention is to provide a method of producing a polypeptide encoded by a polynucleotide comprising that encodes at least a fragment of a leader sequence and a second nucleotide sequence that encodes a polypeptide heterologous to the leader sequence, comprises an amino acid sequence that comprises at least about 70% sequence identity to the leader sequence of Pichia acaciae killer toxin, leader sequence, and suitable for expression thereof, the heterologous polypeptide is produced that is free of additional N-terminal amino acids.
A specific embodiment of the invention is where the heterologous polypeptide is human insulin-like growth factor 1 (IGF-1).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plasmid map of pHIL-A1.
DETAILED DESCRIPTION
Definitions
"Heterologous" means not naturally contiguous. For example, a yeast leader and a human protein are heterologous because the two are not naturally contiguous.
A host cell suitable of "expression of a polynucleotide" is capable of effecting transcription and translation of the polynucleotide to produce the encoded heterologous polypeptide free of additional N-terminal amino acids.
General Methods and Detailed Description
Preferably, polynucleotides of the instant invention are produced by recombinant DNA techniques. The polynucleotide encoding at least a fragment of a leader sequence can be either synthesized or cloned.
The amino acid sequence of the leader sequence comprises at least 70% sequence identity to the leader sequence of the Pichia acaciae killer toxin, described in Bolen et al., Yeast 10: 403-414 (1994) and shown in SEQ ID NO:2. More preferably, the leader sequence comprises at least 80%; even more preferably, at least 90%; more preferably, at least 95% sequence identity to SEQ ID NO:2; most preferably, 100% sequence identity to SEQ ID NO:2.
A full length leader sequence begins at the initiating methionine and ends at the last amino acid residue before the beginning of the encoded mature polypeptide. Amino acid residues can be removed from full length leader to construct leader fragments. These fragments can be tested to determine if they are sufficient for secretion.
Empirical data can be used, for example, to determine if a fragment of a leader sequence is sufficient for se
REFERENCES:
patent: 5324639 (1994-06-01), Brierley et al.
patent: 5330901 (1994-07-01), Prevatt et al.
Bolen et al. Isolation and sequence analysis of a gene from the linear DNA plasmid pPac1-2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis. Yeast vol. 10 pp. 403-414, 1994.
von Heijne The signal peptide. J. Membrane Biology vol. 115 pp. 195-201, 1990.
Koutz et al., "Structural Comparison of the Pichia pastoris Alcohol Oxidase Genes", Yeast 5:167-177 (1989).
Cregg et al., "Functional Characterization of the Two Alcohol Oxidase Genes from the Yeast Pichia pastoris", Molecular and Cellular Biology, vol. 9, No. 3, pp. 1316-1323 (Mar., 1989).
Tschopp et al., "Expression of lacZ gene from Two Methanol-Regulated Promoters in Pichia pastoris", Nucleic Acids Research, vol. 15, No. 9, pp. 3859-3876 (1987).
Sreekrishna et al., "Invertase gene (SUC2) of Saccharomyces cerevisiae as a Dominant Marker for Transformation of Pichia pastoris", Gene, 59:115-125 (1987).
Cregg et al., "Pichia pastoris as a Host System for Transformations", Molecular and Cellular Biology, vol. 5, No. 12, pp. 3376-3385 (Dec. 1985).
Clare et al., "Production of Mouse Epidermal Growth Factor in Yeast: High-Level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies", Gene, 105:205-212 (1991).
Clare et al., "High-Level Expression of Tetanus Toxin Fragment C in Pichia pastoris Strains Containing Multiple Tandem Integrations of the Gene", Bio/Technology, 9:455-460 (May 1991).
Cregg et al., "Use of Site-Specific Recombination to Regenerate Selectable Markers", Mol Gen Genet, 219:320-323 (1989).
Sreekrishna et al., "High-Level Expression, Purification, and Characterization of Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris", Biochemistry, 28:4117-4125 (1989).
Tschopp et al., "High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia pastoris", Bio/Technology, 5:1305-1308 (Dec. 1987).
Bishop Robert J.
Crawford Kenneth
Innis Michael A.
Zaror Isabel
Blackburn Robert P.
Brusca John S.
Chiron Corporation
Guth Joseph H.
Spruill W. Murray
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