US4734362A
Expired / Lapsed
US
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Process for purifying recombinant proteins, and products thereof
Assignee: Cambridge Biotech Corp
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Filing: 1986-09-25
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Granted: 1988-03-29
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Expires: 2006-09-25
Abstract
Abstract The present invention relates to a process for recovering recombinant protein from refractile inclusion bodies of microorganisms which comprise disrupting cells, recovering refractile bodies containing recombinant protein, solubilizing the refractile bodies with a denaturant, protecting sulfhydryl groups of recombinant proteins, converting cationic amino groups of recombinant proteins to anionic carboxylic acid by derivatization with organic cyclic acid anhydrides, and recovering recombinant protein derivatives. The present invention also relates to the production of solid supports coated with recombinant protein recovered by the process of the invention, and the use of said devices for the detection and quantification of antibodies to said recombinant proteins in body fluids.
Inventors
- 👤 Chung-Ho Hung
- 👤 Richard Thorn
- 👤 Charles Riggin
- 👤 Dante Marciani
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 11:27
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K1/1136; C07K16/00; C07K16/08; C07K16/10; C07K16/112; C07K16/114; C08; C08F; C08F8/00; C08F8/42; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54353; G01N33/569; G01N33/56983; G01N33/56988; A; A61; A61K; A61K38/00; C12; C12N; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/14011; C12N2740/14022; C12N2740/16011; C12N2740/16111; C12N2740/16122; Y; Y10; Y10S; Y10S530/00; Y10S530/82; Y10S530/826
Claims Analysis
Total Claims: 38
Claim 1
Independent Claim
54 words
A process for recovering refractile body recombinant protein from microorganisms comprising: (a) disrupting host cells containing said refractile body recombinant protein; (b) solubilizing said refractile body recombinant protein; (c) acid acylating free amino groups contained in said recombinant protein; (d) separating said recombinant protein in N-acid acylated form; and (e) recovering said recombinant protein.
Claim 2
Dependent Claim
54 words
A process for recovering refractile body recombinant protein from microorganisms comprising: (a) disrupting host cells containing said refractile body recombinant protein; (b) solubilizing said refractile body recombinant protein; (c) acid acylating free amino groups contained in said recombinant protein; (d) separating said recombinant protein in N-acid acylated form; and (e) recovering said recombinant protein.
Claim 3
Dependent Claim
26 words
The process of claim 1 wherein sulfhydryl groups, if any, of said recombinant protein are derivatized reversibly or irreversibly prior to acid acylation of amino groups.
Claim 4
Dependent Claim
18 words
The process of claim 1 wherein said recombinant protein comprises a heterologous protein expressed in a host microorganism.
Claim 5
Dependent Claim
12 words
The process of claim 3 wherein the recombinant protein comprises viral antigens.
Claim 6
Dependent Claim
15 words
The process of claim 3 wherein the viral antigen is HTLV-I, HTLV-II, or HTLV-III/LAV antigens.
Claim 7
Dependent Claim
15 words
The process of claim 3 wherein the viral antigen consists of Feline Leukemia Virus antigens.
Claim 8
Dependent Claim
20 words
The process of claim 3 wherein the recombinant protein comprises the expression product of a eukaryotic gene in a microorganism.
Claim 9
Dependent Claim
20 words
The process of claim 3 wherein the recombinant protein comprises the expression product of a prokaryotic gene in a microorganism.
Claim 10
Dependent Claim
14 words
The process of claim 1 wherein said microorganism is bacteria, yeast, fungi, or algae.
Claim 11
Dependent Claim
18 words
The process of claim 1 wherein in step (a) said cells are disrupted utilizing disrupting agents comprising enzymes.
Claim 12
Dependent Claim
24 words
The process of claim 10 wherein said enzymes are selected from the group consisting of Lipozyme, Lysozyme, Lysing Enzyme Mixture, Mutanolysin, Lysostaphin, and DNAse.
Claim 13
Dependent Claim
17 words
The process of claim 1 wherein in said step (a) said cells are disrupted utilizing mechanical means.
Claim 14
Dependent Claim
16 words
The process of claim 1 wherein in said step (b) said solubilizing agent comprises a chemical.
Claim 15
Dependent Claim
13 words
The process of claim 13 wherein said chemical comprises a surface active agent.
Claim 16
Dependent Claim
12 words
The process of claim 13 wherein said chemical comprises a denaturing agent.
Claim 17
Dependent Claim
14 words
The process of claim 15 wherein said denaturing agent is a weak denaturing agent.
Claim 18
Dependent Claim
12 words
The process of claim 16 wherein said weak denaturing agent comprises urea.
Claim 19
Dependent Claim
14 words
The process of claim 15 wherein said denaturing agent is a strong denaturing agent.
Claim 20
Dependent Claim
12 words
The process of claim 18 wherein said strong denaturing agent comprsies guanidine.HCl.
Claim 21
Dependent Claim
12 words
The process of claim 2 wherein said sulfhydryl groups are reversibly derivatized.
Claim 22
Dependent Claim
34 words
The process of claim 20 wherein said sulfhydryl groups are reversibly derivatized by the reversible formation of disulfide bonds utilizing agents selected from the group consisting of cysteine, glutathione, o-iodosobenzoate, sodium tetrathionate, and 5,5'-dithiobis(2-nitrobenzoate).
Claim 23
Dependent Claim
27 words
The process of claim 20 wherein reversal of disulfide formation utilizes reducing agents selected from the group consisting of sodium phosphorothiolate, cysteine, dithiothreitol, 2-mercaptoethanol, and sodium borohydride.
Claim 24
Dependent Claim
11 words
The process of claim 2 wherein sulfhydryl groups are irreversibly alkylated.
Claim 25
Dependent Claim
30 words
The process of claim 23 wherein said sulfhydryl groups are irreversibly alkylated by irreversible alkylating agents selected from the group consisting of iodoacetic acid, iotoacetamide, N-ethylmaleimide, 3-bromopropionic acid, and acrylonitrile.
Claim 26
Dependent Claim
32 words
The process of claim 1 wherein in said claim (d) acid acylation comprises contacting the free amino groups of recombinant proteins with acid acylating reagents at a pH of above about 7.5.
Claim 27
Dependent Claim
15 words
The process of claim 25 wherein said acid acylating reagents are organic cyclic acid anhydrides.
Claim 28
Dependent Claim
14 words
The process of claim 26 wherein said organic cyclic acid anhydride comprises citraconic anhydride.
Claim 29
Dependent Claim
28 words
The process of claim 26 wherein said organic cyclic acid anhydride is maleic anhydride, citraconic anhydride, dimethylmaleic anhydride, exo-3,6-endoxo Δ 4 -tetrahydrophthalic anhydride, or exo-3,6-endoxo-Δ 4 -hexahydrophthalic anhydride.
Claim 30
Dependent Claim
23 words
The process of claim 1 wherein in said claim (e) said recombinant protein in N-acid acylated form is separated from other protein derivitives.
Claim 31
Dependent Claim
20 words
The process of claim 2 wherein said recombinant protein in S-derivatized and N-acylated form is separated from other protein derivatives.
Claim 32
Dependent Claim
14 words
The process of claims 29 or 30 wherein said separation comprises a chromatographic separation.
Claim 33
Dependent Claim
16 words
The process of claim 1 wherein in claim (e) recombinant protein is recovered in native form.
Claim 34
Dependent Claim
14 words
The process of claim 32 wherein sulfhydryl groups are restored to the recombinant protein.
Claim 35
Dependent Claim
20 words
The process of claim 33 wherein sulfhydryl groups are restored to the reduced state by contact with a reducing agent.
Claim 36
Dependent Claim
19 words
The process of claim 34 wherein said reducing agent is sodium phosphorothiolate, 2-mercaptoethanol, dithiothreitol, sodium borohydride or sodium sulfite.
Claim 37
Dependent Claim
15 words
The process of claim 32 wherein free amino groups are restored to the recombinant protein.
Claim 38
Dependent Claim
27 words
The process of claim 36 wherein N-acid acyl groups are removed by contact with acidic buffers of below about pH 6 at appropriate temperatures for appropriate times.
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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) | 1991-03-29 | 1991-09-28 | 1992-03-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-03-29 | 1995-09-28 | 1996-03-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-03-29 | 1999-09-28 | 2000-03-29 | $7700.00 | Window Closed / Lapsed |