US4595658A
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Method for facilitating externalization of proteins synthesized in bacteria
Assignee: Rockefeller University
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Filing: 1982-09-13
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Granted: 1986-06-17
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Expires: 2002-09-13
Abstract
Abstract A method of externalizing proteins from the periplasmic space of gram-negative bacteria and in particular, E. coli and its relatives, comprising utilizing bacteria which have a phage gene, coding for a protein (such as gene III protein) or a mutant bacterial gene (such as a gene coding for a membrane function) which causes perturbation of the outer bacterial membrane resulting in leakage of proteins in the periplasmic space from the cell.
Inventors
- 👤 Norman D. Zinder
- 👤 Peter Model
- 👤 Jef D. Boeke
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 10:56
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/625; C12N15/63; C12N15/70; C07K2319/00; C07K2319/01; C07K2319/035; C12N2795/00; C12N2795/00011; C12N2795/14011; C12N2795/14111; C12N2795/14122; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
159 words
A method for facilitating externalization of proteins transported to the periplasmic space of an E. coli bacterium, comprising: culturing in an appropriate culture medium an E. coli bacterium containing (a) an f1 bacteriophage gene III or gene III fragment coding or gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins transported to the periplasmic space selectively leak from the bacterium and (b) a first DNA sequence coding for the other protein operatively attached to a second DNA sequence coding for a suitable leader capable of mediating transport of the other protein to the periplasmic space, wherein the bacterium is capable of replacing the gene III or gene III fragment and the DNA sequences, and is capable of expressing sufficient amounts of the gene III protein or polypeptide fragment thereof to cause the perturbation, and wherein the other protein is a genetically engineered fusion protein.
Claim 2
Independent Claim
159 words
A method for facilitating externalization of proteins transported to the periplasmic space of an E. coli bacterium, comprising: culturing in an appropriate culture medium an E. coli bacterium containing (a) an f1 bacteriophage gene III or gene III fragment coding or gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins transported to the periplasmic space selectively leak from the bacterium and (b) a first DNA sequence coding for the other protein operatively attached to a second DNA sequence coding for a suitable leader capable of mediating transport of the other protein to the periplasmic space, wherein the bacterium is capable of replacing the gene III or gene III fragment and the DNA sequences, and is capable of expressing sufficient amounts of the gene III protein or polypeptide fragment thereof to cause the perturbation, and wherein the other protein is a genetically engineered fusion protein.
Claim 3
Dependent Claim
31 words
The method of claim 1, wherein the first and second DNA sequences are located on a plasmid and the gene III or gene III fragment is located on the same plasmid.
Claim 4
Dependent Claim
34 words
The method of claim 1, wherein the first DNA sequence coding for the other protein is located on a plasmid and the gene III or gene III fragment is located on a different plasmid.
Claim 5
Dependent Claim
24 words
The method of claim 1, wherein the gene III or gene III fragment serves as the second DNA sequence coding for a suitable leader.
Claim 6
Dependent Claim
28 words
The method of claim 1, wherein the gene III fragment comprises the fragment of phage f1 gene III coding for the amino terminal portion of gene III protein.
Claim 7
Dependent Claim
30 words
The method of claim 1, wherein the gene III fragment comprises the fragment of f1 gene III coding for approximately the 98 amino terminal amino acids of gene III protein.
Claim 8
Dependent Claim
22 words
The method of claim 1, wherein the gene III or gene III fragment is introduced into the bacterium on a recombinant vector.
Claim 9
Dependent Claim
22 words
The method of claim 1, wherein the gene III or gene III fragment is introduced into the bacterium on a recombinant plasmid.
Claim 10
Dependent Claim
24 words
The method of claim 1, wherein the gene III or gene III fragment is introduced into the bacterium by infection with a non-fd phage.
Claim 11
Dependent Claim
24 words
The method of claim 1, wherein the gene III or gene III fragment is introduced into the bacterium by incorporation into the bacterial chromosome.
Claim 12
Independent Claim
88 words
A method for facilitating externalization of proteins from the periplasmic space of an E. coli bacterium comprising: introducing into the bacterium an f1 bacteriophage gene III fragment coding for a gene III polypeptide fragment capable of causing a perturbation of the outer membrane of the bacterium such that other proteins located in the periplasmic space selectively leak from the bacterium, wherein the bacterium is capable of replicating the gene III fragment and is capable of expressing sufficient amounts of the gene III polypeptide fragment to cause the perturbation.
Claim 13
Independent Claim
88 words
A method for facilitating externalization of proteins from the periplasmic space of an E. coli bacterium comprising: introducing into the bacterium an f1 bacteriophage gene III fragment coding for a gene III polypeptide fragment capable of causing a perturbation of the outer membrane of the bacterium such that other proteins located in the periplasmic space selectively leak from the bacterium, wherein the bacterium is capable of replicating the gene III fragment and is capable of expressing sufficient amounts of the gene III polypeptide fragment to cause the perturbation.
Claim 14
Dependent Claim
14 words
The method of claim 11, wherein the other proteins are naturally occuring periplasmic proteins.
Claim 15
Dependent Claim
28 words
The method of claim 11, wherein the gene III fragment comprises the fragment of phage f1 gene III coding for the amino terminal portion of gene III protein.
Claim 16
Dependent Claim
30 words
The method of claim 11, wherein the gene III fragment comprises the fragment of the gene III coding for approximately the 98 amino terminal amino acids of gene III protein.
Claim 17
Dependent Claim
19 words
The method of claim 11, wherein the gene III fragment is introduced into the bacterium on a recombinant vector.
Claim 18
Dependent Claim
19 words
The method of claim 11, wherein the gene III fragment is introduced into the bacterium on a recombinant plasmid.
Claim 19
Dependent Claim
21 words
The method of claim 11, wherein the gene III fragment is introduced into the bacterium by infection with a non-fd phage.
Claim 20
Dependent Claim
21 words
The method of claim 11, wherein the gene III fragment is introduced into the bacterium by incorporation into the bacterial chromosome.
Claim 21
Independent Claim
115 words
A method for isolating proteins externalized from an E. coli bacterium, comprising: (a) culturing in an appropriate culture medium an E. coli bacterium containing (i) an f1 bacteriophage gene III or gene III fragment coding for gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins transported to the periplasmic space selectively leak from the bacterium and (ii) a first DNA sequence coding for said other protein operatively attached to a second DNA sequence coding for a suitable leader capable of mediating transport of said other protein to the periplasmic space; and (b) isolating the leaked proteins from the culture medium.
Claim 22
Independent Claim
115 words
A method for isolating proteins externalized from an E. coli bacterium, comprising: (a) culturing in an appropriate culture medium an E. coli bacterium containing (i) an f1 bacteriophage gene III or gene III fragment coding for gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins transported to the periplasmic space selectively leak from the bacterium and (ii) a first DNA sequence coding for said other protein operatively attached to a second DNA sequence coding for a suitable leader capable of mediating transport of said other protein to the periplasmic space; and (b) isolating the leaked proteins from the culture medium.
Claim 23
Independent Claim
112 words
A method isolating proteins externalized from an E. coli bacterium, comprising: (a) culturing in an appropriate culture medium an E. coli bacterium containing an f1 bacteriophage gene III or gene III fragment coding for gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins located in the periplasmic space selectively leak from the bacterium, wherein said bacterium is capable of replacing said gene III or gene III fragment and is capable of expressing sufficient amounts of said gene III protein or a polypeptide fragment thereof to cause said perturbation; and (b) isolating the leaked proteins from the medium.
Claim 24
Independent Claim
112 words
A method isolating proteins externalized from an E. coli bacterium, comprising: (a) culturing in an appropriate culture medium an E. coli bacterium containing an f1 bacteriophage gene III or gene III fragment coding for gene III protein or a polypeptide fragment thereof capable of causing a perturbation of the outer membrane of the bacterium such that other proteins located in the periplasmic space selectively leak from the bacterium, wherein said bacterium is capable of replacing said gene III or gene III fragment and is capable of expressing sufficient amounts of said gene III protein or a polypeptide fragment thereof to cause said perturbation; and (b) isolating the leaked proteins from the medium.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-06-16 | 1989-12-16 | 1990-06-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-06-16 | 1993-12-16 | 1994-06-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-06-16 | 1997-12-16 | 1998-06-17 | $7700.00 | Window Closed / Lapsed |