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US4731440A
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Method of somatotropin solubilization using dimethylsulfone and naturation
Assignee: Monsanto Technology LLC
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Filing: 1986-08-25
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Granted: 1988-03-15
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Expires: 2006-08-25
Abstract
Abstract A method for solubilization and naturation of somatotropin protein from refractile bodies of host cells is disclosed. The method embraces the discovery that an aqueous solution of urea, or dimethylsulfone, or mixtures of urea and dimethylsulfone, can be effectively used to solubilize refractile bodies containing such somatotropin protein. Once solubilized, somatotropin protein can be natured in an aqueous solution of urea, or dimethylsulfone, or mixtures of urea and dimethylsulfone, by contacting the solution with a mild oxidizing agent for a time sufficient to result in the formation of the disulfide bonds. Naturation can efficiently occur even at high protein concentration, in an impure preparation and in the absence of reducing agent.
Inventors
- 👤 Larry A. Bentle
- 👤 James W. Mitchell
- 👤 Stephen B. Storrs
- 👤 Grant T. Shimamoto
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 15:17
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K14/00; C07K14/435; C07K14/575; C07K14/61; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849; Y10S930/00; Y10S930/01; Y10S930/12
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
42 words
A method for solubilization of somatotropin protein from refractile bodies of a host cell containing said protein which comprises contacting said bodies with an effective amount and concentration of dimethylsulfone or urea-dimethylsulfone at a pH effective to accomplish solubilization of said protein.
Claim 2
Independent Claim
42 words
A method for solubilization of somatotropin protein from refractile bodies of a host cell containing said protein which comprises contacting said bodies with an effective amount and concentration of dimethylsulfone or urea-dimethylsulfone at a pH effective to accomplish solubilization of said protein.
Claim 3
Dependent Claim
26 words
The method of claim 1 in which the solubilization is conducted at a temperature above the freezing point of the solution and below about 25° C.
Claim 4
Dependent Claim
16 words
The method of claim 1 in which solubilization is conducted at a pH above about 9.
Claim 5
Dependent Claim
19 words
The method of claim 1 in which solubilization is conducted at a pH between about 9 and about 12.
Claim 6
Dependent Claim
26 words
The method of claim 1 in which solubilization is conducted at a pH between about 9 and about 12 and a temperature of about 4° C.
Claim 7
Dependent Claim
12 words
The method of claim 1 in which the somatotropin is bovine somatotropin.
Claim 8
Dependent Claim
19 words
The method of claim 6 in which the host cell is a strain of the bacterial species Escherichia coli.
Claim 9
Dependent Claim
41 words
The method of claim 7 in which the dimethylsulfone concentration is between about 0.5 M and 2.4 M; the pH is maintained between about 11 and about 12; and the temperature is maintained between about 5° C. and about 25° C.
Claim 10
Dependent Claim
56 words
The method of claim 1 further comprising naturation of said somatotropin protein which comprises contacting a solution containing a naturation solvent selected from the group consisting of urea, dimethylsulfone and urea-dimethylsulfone, and containing said protein, with a mild oxidizing agent for a time sufficient to form intramolecular disulfide bonds between cysteine residues contained in said somatotropinprotein.
Claim 11
Dependent Claim
17 words
The method of claim 9 in which the somatotropin protein is natured in an aqueous urea solution.
Claim 12
Dependent Claim
23 words
The method of claim 10 in which the naturation is conducted in a urea concentration between about 3 M and about 5 M.
Claim 13
Dependent Claim
17 words
The method of claim 10 in which the naturation is conducted at a pH above about 9.
Claim 14
Dependent Claim
20 words
The method of claim 10 in which the naturation is conducted at a pH between about 9 and about 12.
Claim 15
Dependent Claim
26 words
The method of claim 10 in which the naturation is conducted at a temperature above the freezing point of the solution and below about 25° C.
Claim 16
Dependent Claim
12 words
The method of claim 10 in which the somatotropin is bovine somatotropin.
Claim 17
Dependent Claim
46 words
The method of claim 15 in which the naturation is conducted at a urea concentration between about 2 M and about 5 M; a tempera-ure above the freezing point of the solution and below about 25° C.; and a pH between about 9 and about 12.
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) | 1991-03-15 | 1991-09-14 | 1992-03-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-03-15 | 1995-09-14 | 1996-03-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-03-15 | 1999-09-14 | 2000-03-15 | $7700.00 | Window Closed / Lapsed |