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US4801536A Expired / Lapsed US ⏱️ 0.0 years remaining

Method for producing heterologous proteins

Assignee: Genetics Institute LLC
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Filing: 1987-06-02
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Granted: 1989-01-31
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Expires: 2007-06-02
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Abstract

Abstract This invention concerns a method for producing a heterologous protein in a bacterial host cell such that the protein is exported from the host cell into the culture medium. The method involves culturing in a bacterial culture medium a genetically engineered bacterial strain containing a fusion DNA sequence comprising a first nucleotide sequence encoding at least an N-terminal portion of a flagellin protein and a second nucleotide sequence encoding the heterologous protein. The first nucleotide sequence is linked via its 3' terminus to the 5' terminus of the second nucleotide sequence, and the fusion DNA sequence is itself linked to an expression control sequence. In certain embodiments the first and second nucleotide sequences are linked by means of a linking nucleotide sequence encoding a selectively cleavable polypeptide, In those embodiments the resulting exported fusion protein will contain a selectively cleavable site at which the fusion protein may be selectively cleaved by chemical or enzymatic methods to produce the heterologous protein encoded for by the second nucleotide sequence of the fusion DNA sequence. The heterologous protein may then be separately recovered from any polypeptide fragment of flagellin or other proteinaceous material.

Inventors

  • 👤 Mark L. Stahl
  • 👤 Edward R. LaVallie

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-05 19:41
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C07; C07K; C07K14/00; C07K14/195; C07K14/32; C07K14/435; C07K14/575; C07K14/62; C12N15/87; C12N15/90; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/036; C07K2319/50; C07K2319/70; C07K2319/74; C07K2319/75

Claims Analysis

Total Claims: 10
Claim 1
Independent Claim 122 words
A method for producing a heterologous protein in a bacterial host cell such that the protein is exported from the host cell into the culture medium, the method comprising culturing in a bacterial culture medium a genetically engineered bacterial strain containing a fusion DNA sequence which comprises a first nucleotide sequence encoding at least an N-terminal portion of a flagellin protein native to the bacterial host cell species and a second nucleotide sequence encoding the heterologous protein, said first nucleotide sequence being linked via its 3' terminus to the 5' terminus of the second DNA sequence and said fusion DNA sequence being operatively linked to an expression control sequence and wherein said N-terminal portion results in the export of said heterologous protein.
Claim 2
Independent Claim 122 words
A method for producing a heterologous protein in a bacterial host cell such that the protein is exported from the host cell into the culture medium, the method comprising culturing in a bacterial culture medium a genetically engineered bacterial strain containing a fusion DNA sequence which comprises a first nucleotide sequence encoding at least an N-terminal portion of a flagellin protein native to the bacterial host cell species and a second nucleotide sequence encoding the heterologous protein, said first nucleotide sequence being linked via its 3' terminus to the 5' terminus of the second DNA sequence and said fusion DNA sequence being operatively linked to an expression control sequence and wherein said N-terminal portion results in the export of said heterologous protein.
Claim 3
Dependent Claim 17 words
A method according to claim 1 which further comprises recovering the exported protein from the culture medium.
Claim 4
Dependent Claim 41 words
A method according to claim 1, wherein the first and second nucleotide sequences of the fusion DNA sequence are linked by a linking nucleotide sequence which encodes a selectably cleavable polypeptide such that the exported protein contains a selectably cleavable site.
Claim 5
Dependent Claim 35 words
A method according to claim 3 which further comprises cleaving the exported protein at the selectably cleavable site to produce the heterologous protein encoded for by the second nucleotide sequence of the fusion DNA sequence.
Claim 6
Dependent Claim 23 words
A method according to claim 4 which further comprises recovering the heterologous protein from any polypeptide fragment of flagellin or other proteinaceous material.
Claim 7
Dependent Claim 27 words
A method according to claim 1, wherein the engineered bacterial cells are cultured in the presence of a substance which represses the production or export of proteases.
Claim 8
Dependent Claim 20 words
A method according to claim 1, wherein the fusion DNA sequence is integrated into the chromosome of the host cell.
Claim 9
Dependent Claim 21 words
A method according to claim 1, wherein the fusion DNA sequence is contained in an extrachromosomal plasmid within the host cell.
Claim 10
Dependent Claim 18 words
A method according to claim 2, wherein the exported protein is recovered by immunoaffinity chromatography using anti-flagellin antibody.

References Cited (Backward Citations)

US4338397A Cited
EP0237045A2 Cited

USPTO Annuity & Maintenance Windows

Annuity Stage Window Opens Payment Deadline Surcharge Period Until Estimated Fee Status
3.5 Years (Year 4) 1992-01-31 1992-08-01 1993-01-31 $2000.00 Window Closed / Lapsed
7.5 Years (Year 8) 1996-01-31 1996-08-01 1997-01-31 $3760.00 Window Closed / Lapsed
11.5 Years (Year 12) 2000-01-31 2000-08-01 2001-01-31 $7700.00 Window Closed / Lapsed

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