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US4751180A Expired / Lapsed US ⏱️ 0.0 years remaining

Expression using fused genes providing for protein product

Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1986-03-28
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Granted: 1988-06-14
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Expires: 2006-03-28
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Abstract

Abstract Novel methods and compositions are provided for enhanced yield of heterologous proteins in fungi. The method and compositions involve employing fusion sequences involving a sequence encoding a heterologous product produced in relatively large amount as a stable polypeptide in the host fused to a second sequence in open reading frame with the prior sequence coding for a different heterologous polypeptide, where the two polypeptides are joined by a selectively cleavable linkage. In particular, a sequence coding for superoxide dismutase is joined to another polypeptide of interest at either terminus of the superoxide dismutase in a yeast expression vector under transcriptional control of an active promoter and the vector introduced into a yeast host and the host grown. High yields of the fusion product are obtained in this manner, where the fusion product can be selectively cleaved so as to produce both the superoxide dismutase and the other polypeptide in high yield. The S. cerevisiae strain 2150-2-3 (pYASI1) was deposited at the A.T.C.C. on Feb. 27, 1985 and given accession No. 20745. The S. cerevisiae strain AB110 (pYLUIGF2-14) was deposited at the A.T.C.C. on Mar. 19, 1986 and given accession No. 20796.

Inventors

  • 👤 Lawrence S. Cousens
  • 👤 Patricia A. Tekamp-Olson
  • 👤 Jeffrey R. Shuster
  • 👤 James P. Merryweather

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-03 23:16
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C07K14/435; C07K14/575; C07K14/62; C07K14/65; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N9/00; C12N9/0004; C12N9/0089; C12Y; C12Y115/00; C12Y115/01; C12Y115/01001; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/50; C07K2319/70; C07K2319/74; C07K2319/75; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/16011; C12N2740/16211; C12N2740/16222; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/31

Claims Analysis

Total Claims: 25
Claim 1
Independent Claim 96 words
In a method for preparing a polypeptide in a cellular host, where the polypeptide is heterologous to the host and may be produced in low percentage amounts of total protein, the improvement which comprises: joining an open reading frame DNA sequence coding for said polypeptide with a second open reading frame DNA sequence coding for heterologous superoxide dismutase to form a fusion polypeptide; introducing the sequence coding for said fusion polypeptide under conditions for expression in said host, whereby said fusion polypeptide is expressed; and isolating said fusion polypeptide to provide the polypeptide in high yield.
Claim 2
Dependent Claim 96 words
In a method for preparing a polypeptide in a cellular host, where the polypeptide is heterologous to the host and may be produced in low percentage amounts of total protein, the improvement which comprises: joining an open reading frame DNA sequence coding for said polypeptide with a second open reading frame DNA sequence coding for heterologous superoxide dismutase to form a fusion polypeptide; introducing the sequence coding for said fusion polypeptide under conditions for expression in said host, whereby said fusion polypeptide is expressed; and isolating said fusion polypeptide to provide the polypeptide in high yield.
Claim 3
Dependent Claim 15 words
A method according to claim 1, wherein said superoxide dismutase is human superoxide dismutase (hSOD).
Claim 4
Dependent Claim 11 words
A method according to claim 2, wherein said host is eukaryotic.
Claim 5
Dependent Claim 12 words
A method according to claim 3, wherein said eukaryotic host is yeast.
Claim 6
Dependent Claim 13 words
A method according to claim 2 wherein said polypeptide is a mammalian polypeptide.
Claim 7
Dependent Claim 22 words
A method according to claim 5, wherein said mammalian polypeptide is 3' to said hSOD coding sequence in the direction of transcription.
Claim 8
Dependent Claim 24 words
A method according to claim 5, wherein said mammalian polypeptide is 5' to said DNA sequence coding for SOD in the direction of transcription.
Claim 9
Dependent Claim 18 words
A method according to claim 5, wherein said conditions for expression include an inducible transcriptional initiation regulatory region.
Claim 10
Dependent Claim 34 words
A method according to claim 8, where said transcriptional intiation regulator region consists essentially of, reading in a 5' to 3' direction, a yeast glycolytic enzyme promoter region and adjacent alcohol dehydrogenase-2 control region.
Claim 11
Dependent Claim 11 words
A method according to claim 2, wherein said host is prokaryotic.
Claim 12
Dependent Claim 13 words
A method according to claim 10, wherein said prokaryotic host is E. coli.
Claim 13
Dependent Claim 50 words
A DNA sequence coding for superoxide dismutase joined to a DNA sequence coding for a mammalian polypeptide, where the two coding sequences are joined by bases coding for amino acids defining a selectively cleavable link having at least one amino acid, wherein said link provides for a selectively cleavable site.
Claim 14
Dependent Claim 50 words
A DNA sequence coding for superoxide dismutase joined to a DNA sequence coding for a mammalian polypeptide, where the two coding sequences are joined by bases coding for amino acids defining a selectively cleavable link having at least one amino acid, wherein said link provides for a selectively cleavable site.
Claim 15
Dependent Claim 14 words
A DNA sequence according to claim 12, wherein said cleavable link codes for methionine.
Claim 16
Dependent Claim 13 words
A DNA sequence according to claim 12, wherein said cleavable link is Lys-Arg.
Claim 17
Dependent Claim 15 words
A DNA sequence according to claim 12, wherein said cleavable link is (ASp) 4 Lys.
Claim 18
Dependent Claim 15 words
A DN.A sequence according to claim 12, wherein said cleavable link includes hinge amino acids.
Claim 19
Dependent Claim 16 words
A DNA sequence according to claim 12, wherein said cleavable link is an enzymatically removable link.
Claim 20
Dependent Claim 23 words
An expression sequence including, in the direction of transcription, an inducible transcriptional initiation regulatory region and a DNA sequence according to claim 10.
Claim 21
Dependent Claim 34 words
A DNA sequence coding for superoxide dismutase joined to a DNA sequence coding for a mammalian polypeptide, where the two coding sequences are joined by bases coding for amino acids defining a hinge sequence.
Claim 22
Dependent Claim 34 words
A DNA sequence coding for superoxide dismutase joined to a DNA sequence coding for a mammalian polypeptide, where the two coding sequences are joined by bases coding for amino acids defining a hinge sequence.
Claim 23
Dependent Claim 18 words
The DNA sequence according to claim 19 wherein said hinge sequence is from one to fifteen amino acids.
Claim 24
Dependent Claim 30 words
The DNA sequence according to claim 20 wherein said hinge sequence is composed of nonpolar amino acids consisting of the group selected from glycine, alanine, proline, valine, isoleucine and leucine.
Claim 25
Dependent Claim 28 words
The DNA sequence according to claim 20 wherein said hinge sequence is composed of polar amino acids consisting of the group selected from asparagine, glutamine, serine and threonine.

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USPTO Annuity & Maintenance Windows

Annuity Stage Window Opens Payment Deadline Surcharge Period Until Estimated Fee Status
3.5 Years (Year 4) 1991-06-14 1991-12-14 1992-06-14 $2000.00 Window Closed / Lapsed
7.5 Years (Year 8) 1995-06-14 1995-12-14 1996-06-14 $3760.00 Window Closed / Lapsed
11.5 Years (Year 12) 1999-06-14 1999-12-14 2000-06-14 $7700.00 Window Closed / Lapsed

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