US5480794A
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Overproduction and purification of soluble PHA synthase
Assignee: Yield10 Bioscience Inc
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Filing: 1994-08-12
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Granted: 1996-01-02
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Expires: 2014-08-12
Abstract
Abstract Purified, soluble recombinant bacterial long chain and short chain PHA synthases are described, with methods and materials for overexpression and purification.
Inventors
- 👤 Oliver P. Peoples
- 👤 Tillman U. Gerngross
- 👤 Anthony J. Sinskey
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 08:00
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/93; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12N9/0004; C12N9/0006; C12N9/10; C12N9/1025; C12N9/1029; C12P; C12P7/00; C12P7/62; C12P7/625
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
52 words
A method for the overproduction and purification of soluble polyhydroxyalkanoate (PHA) synthase expressed from an isolated structural gene comprising expressing the isolated structural gene encoding the synthase in a vector in an expression system not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants.
Claim 2
Independent Claim
52 words
A method for the overproduction and purification of soluble polyhydroxyalkanoate (PHA) synthase expressed from an isolated structural gene comprising expressing the isolated structural gene encoding the synthase in a vector in an expression system not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants.
Claim 3
Dependent Claim
20 words
The method of claim 1 further comprising placing the PHA synthase gene under the transcriptional control of a regulatable promoter.
Claim 4
Dependent Claim
42 words
The method of claim 2 wherein the promoter is selected from the group consisting of an IPTG inducible promoter, lacZ, tac, trc, the trp promoter, the temperature inducible phage promoter lambda P L , and a phage T7 promoter/T7 RNA polymerase system.
Claim 5
Dependent Claim
14 words
The method of claim 1 where the PHA synthase is a polyhydroxyalkamoate (SCPHA) synthase.
Claim 6
Dependent Claim
16 words
The method of claim 1 where the PHA synthase is a SCPHA synthase from Alcaligenes eutrophus.
Claim 7
Dependent Claim
14 words
The method of claim 1 where the PHA synthase is a polyhydroxyalkanoate (LCPHA) synthase.
Claim 8
Dependent Claim
19 words
The method of claim 1 where the PHA synthase is a LCPHA synthase encoded by a gene from Pseudomonas.
Claim 9
Dependent Claim
37 words
The method of claim 1 where the PHA synthase is overproduced in a host selected from the group including bacteria, yeast, fungi and plants and the vector comprises a ribosome binding site enhancing expression in the host.
Claim 10
Dependent Claim
15 words
The method of claim 1 where the PHA synthase is overproduced in a bacterial host.
Claim 11
Dependent Claim
14 words
The method of claim 1 where the PHA synthase is overproduced in Escherichia coli.
Claim 12
Dependent Claim
19 words
The method of claim 10 where the PHA synthase is overproduced in a protease deficient strain of Escherichia coli.
Claim 13
Dependent Claim
18 words
The method of claim 11 where the PHA synthase is expressed in an ompT deficient Escherichia coli strain.
Claim 14
Dependent Claim
28 words
The method of claim 10 where the PHA synthase is engineered so as to remove cleavage sites that are normally cleaved by proteases in the naturally occurring synthase.
Claim 15
Dependent Claim
18 words
The method of claim 1 further comprising purifying the PHA synthase by chromatography using buffer solutions containing detergents.
Claim 16
Dependent Claim
18 words
The method of claim 14 where the PHA synthase is purified by chromatography using buffer solutions containing 6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside.
Claim 17
Dependent Claim
14 words
The method of claim 15 wherein the synthase is purified by chromatography on hydroxyapatite.
Claim 18
Dependent Claim
58 words
An expression system for the overproduction and purification of soluble PHA synthase expressed from an isolated structural gene comprising a host not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants, and the isolated structural gene encoding PHA synthase in a vector for expression of the synthase in the host.
Claim 19
Dependent Claim
58 words
An expression system for the overproduction and purification of soluble PHA synthase expressed from an isolated structural gene comprising a host not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants, and the isolated structural gene encoding PHA synthase in a vector for expression of the synthase in the host.
Claim 20
Dependent Claim
19 words
The system of claim 17 wherein the PHA synthase gene is under the transcriptional control of a regulatable promoter.
Claim 21
Dependent Claim
21 words
The system of claim 17 where the PHA synthase is selected from the group consisting of SCPHA synthase and LCPHA synthase.
Claim 22
Dependent Claim
15 words
The system of claim 17 where the PHA synthase is overproduced in a bacterial host.
Claim 23
Dependent Claim
14 words
The system of claim 17 where the PHA synthase is overproduced in Escherichia coli.
Claim 24
Dependent Claim
19 words
The system of claim 17 where the PHA synthase is overproduced in a protease deficient strain of Escherichia coli.
Claim 25
Dependent Claim
28 words
The method of claim 17 where the PHA synthase is engineered so as to remove cleavage sites that are normally cleaved by proteases in the naturally occurring synthase.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-01-01 | 1999-07-03 | 2000-01-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-01-01 | 2003-07-03 | 2004-01-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-01-01 | 2007-07-03 | 2008-01-02 | $7700.00 | Window Closed / Lapsed |