EP0116201A1
Expired / Lapsed
EP
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Secretory expression in eukaryotes
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1983-10-26
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Granted: —
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Expires: 2003-10-26
Abstract
Abstract Methods and compositions are provided for producing polypeptide sequences in high yield by employing DNA constructs, wherein the DNA sequence encoding for the polypeptide of interest is preceded by a leader sequence and processing sequence for secreting and processing said polypeptide. In this manner, the mature polypeptide of interest may be isolated from the nutrient medium substantially free of major amounts of other proteins and cellular debris. The yeast strain S. cerevisiae AB103 (pYEGF8) was deposited on January 5, 1983, at the A.T.C.C. and given accession No. 20658. The plasmid pYaEGF23 (pAB114-pC1/1) was deposited at the A.T.C.C. on August 12,1983, and given Accession No. 40079.
Inventors
- 👤 Anthony John Brake
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 14:55
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/485; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/625; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C07K2319/00; C07K2319/01; C07K2319/036; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
43 words
A DNA construct encoding a pre-pro-polypeptide, said DNA construct encoding pre-pro-polypeptide comprising a yeast leader sequence, processing signals for processing the pre-pro-polypeptide to a mature polypeptide and a gene encoding a polypeptide other than the wild type gene associated with said leader sequence.
Claim 2
Dependent Claim
43 words
A DNA construct encoding a pre-pro-polypeptide, said DNA construct encoding pre-pro-polypeptide comprising a yeast leader sequence, processing signals for processing the pre-pro-polypeptide to a mature polypeptide and a gene encoding a polypeptide other than the wild type gene associated with said leader sequence.
Claim 3
Dependent Claim
28 words
A DNA construct according to Claim 1, including at the 5' end of the sequence a yeast promoter and wherein said gene is heterologous to said yeast host.
Claim 4
Dependent Claim
41 words
A DNA construct according to Claim 2, wherein said yeast promoter is the a-factor promoter and said yeast leader is a leader sequence encoding for at least a major portion of the a-factor leader and is capable of providing for secretion.
Claim 5
Dependent Claim
14 words
A DNA construct according to Claim 2, wherein said gene is a mammalian gene.
Claim 6
Dependent Claim
75 words
A DNA construct comprising a sequence of the following formula: wherein: L is a leader sequence recognized by yeast for secretion; R is a codon coding for arginine or lysine: r is an integer of from 2 to 4; X is any nucleotide; Y is guanosine or cytosine; y is an integer of from about 1 to 10; G ene * is a gene foreign to yeast; and n is 0 or 1 to 4.
Claim 7
Dependent Claim
75 words
A DNA construct comprising a sequence of the following formula: wherein: L is a leader sequence recognized by yeast for secretion; R is a codon coding for arginine or lysine: r is an integer of from 2 to 4; X is any nucleotide; Y is guanosine or cytosine; y is an integer of from about 1 to 10; G ene * is a gene foreign to yeast; and n is 0 or 1 to 4.
Claim 8
Dependent Claim
35 words
A DNA construct according to Claim 5, wherein n is 0 to 4 and the nucleotides of said Gene * proximal to R at least in part define a recognition site for a restriction endonuclease.
Claim 9
Dependent Claim
16 words
A DNA construct according to Claim 6, wherein said leader sequence is the a-factor leader sequence.
Claim 10
Dependent Claim
11 words
A DNA construct according to Claim 7, wherein n is 0.
Claim 11
Dependent Claim
141 words
A DNA construct of the formula: wherein: Tr is a sequence having transcriptional and translational regulatory signals for initiation and processing of transcription and translation, wherein said regulatory signals are recognized by yeast; L is a leader sequence for secretion by yeast; R and S are codons expressing arginine and lysine; X is any nucleotide; Y is cytosine or guanosine; y is an integer of from 1 to 4; n' is a whole number of from 0 to 4; W is a deoxyribosyl-3' group or when n' is other than 0, one or more nucleotides which by themselves or together with the hexanucleotide in the parenthesis define a restriction site; Gene * either by itself or taken together with W defines a polypeptide sequence foreign to yeast; and d is 0 or 1, being 1, when y is greater than 1.
Claim 12
Dependent Claim
141 words
A DNA construct of the formula: wherein: Tr is a sequence having transcriptional and translational regulatory signals for initiation and processing of transcription and translation, wherein said regulatory signals are recognized by yeast; L is a leader sequence for secretion by yeast; R and S are codons expressing arginine and lysine; X is any nucleotide; Y is cytosine or guanosine; y is an integer of from 1 to 4; n' is a whole number of from 0 to 4; W is a deoxyribosyl-3' group or when n' is other than 0, one or more nucleotides which by themselves or together with the hexanucleotide in the parenthesis define a restriction site; Gene * either by itself or taken together with W defines a polypeptide sequence foreign to yeast; and d is 0 or 1, being 1, when y is greater than 1.
Claim 13
Dependent Claim
36 words
A DNA construct according to Claim 9, wherein Tr is a sequence defining the regulatory signals for a-factor, d is 1 and Gene * and W are taken together to define a polypeptide foreign to yeast.
Claim 14
Dependent Claim
11 words
A DNA construct according to Claim 9 wherein n' is 0.
Claim 15
Dependent Claim
15 words
A DNA construct according to Claim 11, wherein said polypeptide product is a mammalian polypeptide.
Claim 16
Dependent Claim
115 words
A DNA construct comprising a sequence of the formula: wherein: Tr is a sequence defining transcriptional and translational regulatory signals for initiation and processing of transcription and translation recognized by yeast; a is 0 or 1; L is a leader sequence recognized by yeast; R and S are codons encoding for lysine and arginine; X is any nucleotide; Y is cytosine or guanosine; n" is 2 to 4; the nucleotides in the broken box indicate the nucleotides which are complementary to the overhang of the non-coding chain to define a HindIII restriction site. 14. A DNA construct according to Claim 13, where Tr is a sequence defining the transcriptional and translational regulatory signals of a-factor.
Claim 17
Dependent Claim
136 words
An expression episomal element comprising a replication system for providing stable maintenance in yeast and a sequence of the formula: wherein: Tr is a sequence defining transcriptional and translational regulatory signals for initiation and processing of transcription and translation in yeast; L is a leader sequence recognized by yeast for secretion; R is a codon defining arginine or lysine; r' is a whole number in the range of 2 to 4; X is any nucleotide; Y is cytosine or guanosine; n' is a whole number in the range of 0 to 4; W is a nucleotide sequence of at least 1 nucleotide, which by itself or when n' is other than 0, in conjunction with nucleotides in the parenthesis defines a restriction site; Te is a sequence defining a terminator balanced with said transcriptional initiator sequence.
Claim 18
Dependent Claim
136 words
An expression episomal element comprising a replication system for providing stable maintenance in yeast and a sequence of the formula: wherein: Tr is a sequence defining transcriptional and translational regulatory signals for initiation and processing of transcription and translation in yeast; L is a leader sequence recognized by yeast for secretion; R is a codon defining arginine or lysine; r' is a whole number in the range of 2 to 4; X is any nucleotide; Y is cytosine or guanosine; n' is a whole number in the range of 0 to 4; W is a nucleotide sequence of at least 1 nucleotide, which by itself or when n' is other than 0, in conjunction with nucleotides in the parenthesis defines a restriction site; Te is a sequence defining a terminator balanced with said transcriptional initiator sequence.
Claim 19
Dependent Claim
20 words
An expression episomal element according to Claim 15 wherein Tr is derived from a-factor and n' is 2 to 3.
Claim 20
Dependent Claim
18 words
An expression episomal element according to Claim 14, wherein Tr is derived from a-factor and n' is 0.
Claim 21
Dependent Claim
28 words
An episomal expression vector according to Claim 17, having a gene foreign to yeast intermediate R and Te and in reading frame with the initiation codon of L.
Claim 22
Dependent Claim
15 words
An episomal expression element according to Claim 18, wherein said gene is a mammalian gene.
Claim 23
Dependent Claim
16 words
An episomal element according to Claim 19, wherein said mammalian gene is human epidermal growth factor.
Claim 24
Dependent Claim
32 words
An episomal expression vector according to Claim 16, having a gene foreign to yeast intermediate the nucleotides in the parentheses and Te and in reading frame with the initiation codon of L.
Claim 25
Dependent Claim
52 words
A method for producing a polypeptide foreign to yeast and having such polypeptide secreted into the culture medium, said method comprising: growing yeast containing an episomal expression elements according to Claim 16, whereby the encoding sequences are expressed to produce a pre-pro-polypeptide; and said pre-pro-polypeptide is at least partially processed and secreted.
Claim 26
Dependent Claim
32 words
An episomal expression vector according to Claim 17, having a gene foreign to yeast intermediate the nucleotides in the parentheses and Te and in reading frame with the initiation codon of L.
Claim 27
Dependent Claim
64 words
A method for producing a polypeptide foreign to yeast and having such polypeptide secreted into the culture medium, said method comprising: growing yeast mutants containing an episomal expression element according to Claim 16, wherein said mutant permits external regulation of expression, whereby the encoding sequences are expressed to produce a pre-pro-polypeptide under permissive conditions; and said pre-pro-polypeptide is at least partially processed and secreted.
Claim 28
Dependent Claim
15 words
A method according to Claim 24, wherein said mutant yeast is a temperature-sensitive sir mutant.
Claim 29
Dependent Claim
64 words
A method for producing a polypeptide foreign to yeast and having such polypeptide secreted into the culture medium, said method comprising: growing yeast mutants containing an episomal expression element according to Claim 17, wherein said mutant permits external regulation of expression, whereby the encoding sequences are expressed to produce a pre-pro-polypeptide under permissive conditions; and said pre-pro-polypeptide is at least partially processed and secreted.
Claim 30
Dependent Claim
15 words
A method according to Claim 26, wherein said mutant yeast is a temperature-sensitive sir mutant.
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