US4677196A
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Purification and activation of proteins from insoluble inclusion bodies
Assignee: International Minerals and Chemical Corp
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Filing: 1985-09-06
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Granted: 1987-06-30
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Expires: 2005-09-06
Abstract
Abstract Purification and activation of proteins produced in transformant microorganisms as insoluble, biologically inactive inclusion bodies can be effected by solubilizing the inclusion bodies in SDS; dialyzing or refrigerating the protein solution to remove a portion of the SDS; chromatographing the protein solution on an ion-retardation resin to remove the remaining SDS from the protein; and chromatographing the protein solution thus obtained on an anion-exchange resin.
Inventors
- 👤 Steven K. Rausch
- 👤 Hsi Meng
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 14:15
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/61; C07K1/00; C07K1/14; C07K1/36; Y; Y10; Y10S; Y10S530/00; Y10S530/82
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
90 words
A method for purifying and recovering as biologically active a protein which is produced as an insoluble, biologically inactive inclusion body in a transformant microorganism which comprises: (a) extracting the inclusion bodies into a buffered solution of sodium dodecyl sulfate to solubilize the protein; (b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein and recover a biologically active protein; and (c) chromatographing the biologically active protein solution from step (b) on an anion-exchange resin thereby recovering a purified biologically active protein.
Claim 2
Independent Claim
90 words
A method for purifying and recovering as biologically active a protein which is produced as an insoluble, biologically inactive inclusion body in a transformant microorganism which comprises: (a) extracting the inclusion bodies into a buffered solution of sodium dodecyl sulfate to solubilize the protein; (b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein and recover a biologically active protein; and (c) chromatographing the biologically active protein solution from step (b) on an anion-exchange resin thereby recovering a purified biologically active protein.
Claim 3
Dependent Claim
30 words
A method as claimed in claim 1, which further comprises removing a portion of the sodium dodecyl sulfate from the protein solution prior to chromatographing it on the ion-retardation resin.
Claim 4
Dependent Claim
27 words
A method as claimed in claim 2, wherein the SDS is partially removed, prior to ion-retardation chromatography, by dialyzing the protein solution against a SDS-free buffer solution.
Claim 5
Dependent Claim
25 words
A method as claimed in claim 3, wherein about 40% to about 70% of the SDS is removed prior to chromatography on the ion-retardation resin.
Claim 6
Dependent Claim
34 words
A method as claimed in claim 2, wherein the SDS is removed, prior to ion-retardation chromatography, by cooling the protein solution to temperature between the freezing point of the solution and about 5° C.
Claim 7
Dependent Claim
25 words
A method as claimed in claim 5, wherein about 40% to about 70% of the SDS is removed prior to chromatography on the ion-retardation resin.
Claim 8
Dependent Claim
15 words
A method as claimed in claim 1, wherein the protein is an animal growth hormone.
Claim 9
Dependent Claim
22 words
A method as claimed in claim 7, wherein the animal growth hormone is bovine growth hormone or a fragment or analog thereof.
Claim 10
Dependent Claim
22 words
A method as claimed in claim 7, wherein the animal growth hormone is porcine growth hormone or a fragment or analog thereof.
Claim 11
Dependent Claim
26 words
A method as claimed in claim 1, wherein the inclusion bodies are extracted into a buffered solution containing from about 0.1% to 5.0% sodium dodecyl sulfate.
Claim 12
Dependent Claim
23 words
A method as claimed in claim 1, wherein the inclusion bodies are extracted into a buffered solution containing about 1% sodium dodecyl sulfate.
Claim 13
Dependent Claim
23 words
A method as claimed in claim 1, wherein the sodium dodecyl sulfate solution has a pH from about pH 8.5 to pH 11.
Claim 14
Dependent Claim
13 words
A method as claimed in claim 1, wherein the ion-retardation resin is AG11A8.
Claim 15
Dependent Claim
13 words
A method as claimed in claim 1, wherein the anion-exchange resin is DE-53.
Claim 16
Dependent Claim
12 words
A method as claimed in claim 8, wherein the protein is Δ9(Ser)bGH.
Claim 17
Dependent Claim
13 words
A method as claimed in claim 9, wherein the protein is Δ7 pGH.
Claim 18
Dependent Claim
29 words
A method as claimed in claim 1, wherein said buffered solution is selected from the group consisting of 0.02 M to 0.1 M ethanolamine-HCl, carbonate-bicarbonate, and borate buffer solutions.
Claim 19
Independent Claim
129 words
A method for purifying and recovering as biologically active a protein which is produced as an insoluble, biologically inactive inclusion body in a transformant microorganism which comprises: (a) extracting the inclusion bodies into a buffered solution of from about 0.1% to about 5.0% sodium dodecyl sulfate to solubilize the protein, said buffer solution selected from the group consisting of 0.02 M to 0.1 M ethanolamine-HCl, carbonate-bicarbonate, and borate buffer solutions, said buffered solutions having a pH from about pH 8.5 to pH 11; (b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein and recover a biologically active protein; and (c) chromatographing the biologically active protein solution from step (b) on an anion-exchange resin thereby recovering a purified biologically active protein.
Claim 20
Independent Claim
129 words
A method for purifying and recovering as biologically active a protein which is produced as an insoluble, biologically inactive inclusion body in a transformant microorganism which comprises: (a) extracting the inclusion bodies into a buffered solution of from about 0.1% to about 5.0% sodium dodecyl sulfate to solubilize the protein, said buffer solution selected from the group consisting of 0.02 M to 0.1 M ethanolamine-HCl, carbonate-bicarbonate, and borate buffer solutions, said buffered solutions having a pH from about pH 8.5 to pH 11; (b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein and recover a biologically active protein; and (c) chromatographing the biologically active protein solution from step (b) on an anion-exchange resin thereby recovering a purified biologically active protein.
Claim 21
Dependent Claim
23 words
A method as claimed in claim 18, wherein the inclusion bodies are extracted into a buffered solution containing about 1% sodium dodecyl sulfate.
Claim 22
Dependent Claim
27 words
A method as claimed in claim 18, wherein the SDS is partially removed, prior to ion-retardation chromatography, by dialyzing the protein solution against a SDS-free buffer solution.
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) | 1990-06-29 | 1990-12-29 | 1991-06-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-29 | 1994-12-29 | 1995-06-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-29 | 1998-12-29 | 1999-06-30 | $7700.00 | Window Closed / Lapsed |