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US4943527A Expired / Lapsed US ⏱️ 0.0 years remaining

Mature apoai protein production under serum free culturing conditions

Assignee: Scios LLC
•
Filing: 1986-02-27
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Granted: 1990-07-24
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Expires: 2006-02-27
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Abstract

Abstract A method of producing a purified lipid-binding peptide which can bind to phospholipids at one or more amphipatic alpha-helical peptide regions. The method includes providing a gene coding for the peptide, and introducing the gene in expressible, heterologous form in a suitable expression system capable of synthesizing a mixture of peptides which includes the lipid-binding peptide. Addition of either endogenous or exogenous lipids to the peptide mixture forms a low-density lipopeptide complex composed of lipid and the lipid-binding peptide, and this complex can be separated easily from nonlipid-binding peptides in the peptide on the basis of its size and/or density. The method is intended particularly for scaled-up production of purified human apolipoproteins and their alpha-helical lipid-binding regions. Also disclosed are related methods for producing recombinant apolipoproteins, therapeutic lipopeptide compositions, and a stabilized lipid emulsion for nutritional therapy. Further disclosed are methods for expressing apolipoproteins or lipid-binding segments thereof in bacterial, yeast and mammalian cell expression systems, and methods for purifying lipid binding proteins, including fused recombinant proteins.

Inventors

  • 👤 Andrew A. Protter
  • 👤 Jean-Louis Vigne
  • 👤 Joanne B. Mallory
  • 👤 Karen D. Talmadge
  • 👤 John P. Kane

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-07 14:17
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/775; A; A61; A61K; A61K31/00; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/63; C12N15/79; C12N15/85; C12P; C12P21/00; C12P21/02; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/70; C07K2319/74; C07K2319/75

Claims Analysis

Total Claims: 6
Claim 1
Independent Claim 86 words
A method of producing purified human apoAI comprising: providing a Chinese hamster ovary (CHO) cell transformed with an expression vector comprising a regulatable human metallothionein II gene promoter, and an expressible, heterologous gene coding for prepro apoAI, culturing the cell in a serum-free medium under promoter-induction conditions wherein a mixture of extracellular proteins containing mature apoAI is produced, forming a lipoprotein complex composed of endogenous CHO cell lipid particles and associated apoAI, and separating the lipoprotein complex from nonlipid-binding proteins in the extracellular medium by floatation.
Claim 2
Independent Claim 86 words
A method of producing purified human apoAI comprising: providing a Chinese hamster ovary (CHO) cell transformed with an expression vector comprising a regulatable human metallothionein II gene promoter, and an expressible, heterologous gene coding for prepro apoAI, culturing the cell in a serum-free medium under promoter-induction conditions wherein a mixture of extracellular proteins containing mature apoAI is produced, forming a lipoprotein complex composed of endogenous CHO cell lipid particles and associated apoAI, and separating the lipoprotein complex from nonlipid-binding proteins in the extracellular medium by floatation.
Claim 3
Independent Claim 67 words
A method of producing human apoAI comprising: providing a suitable mammalian expression system capable of synthesizing a protein mixture which includes an expressed heterologous gene product wherein the expression system comprises a Chinese hamster ovary (CHO) cell transformed with an expressible, heterologous gene coding for prepro apoAI, and culturing the CHO cell in a serum-free medium under conditions which permit the production of mature human apoAI protein.
Claim 4
Independent Claim 67 words
A method of producing human apoAI comprising: providing a suitable mammalian expression system capable of synthesizing a protein mixture which includes an expressed heterologous gene product wherein the expression system comprises a Chinese hamster ovary (CHO) cell transformed with an expressible, heterologous gene coding for prepro apoAI, and culturing the CHO cell in a serum-free medium under conditions which permit the production of mature human apoAI protein.
Claim 5
Dependent Claim 25 words
The method of claim 2, wherein the gene is introduced in an expression vector under the control therein of a regulatable human metallothionein-II gene promoter.
Claim 6
Dependent Claim 24 words
The method of claim 3, wherein the CHO cells include a clonal strain of cells which are selected for high-level expression of the gene.

References Cited (Backward Citations)

US4349629A Cited
US4399216A Cited
US4579821A Cited
US4546082A Cited

USPTO Annuity & Maintenance Windows

Annuity Stage Window Opens Payment Deadline Surcharge Period Until Estimated Fee Status
3.5 Years (Year 4) 1993-07-23 1994-01-22 1994-07-24 $2000.00 Window Closed / Lapsed
7.5 Years (Year 8) 1997-07-23 1998-01-22 1998-07-24 $3760.00 Window Closed / Lapsed
11.5 Years (Year 12) 2001-07-23 2002-01-22 2002-07-24 $7700.00 Window Closed / Lapsed

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