US4694073A
Expired / Lapsed
US
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Method of somatotropin solubilization and naturation using dimethylsulfoxide
Assignee: Monsanto Technology LLC
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Filing: 1986-08-25
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Granted: 1987-09-15
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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 01:33
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K14/00; C07K14/435; C07K14/575; C07K14/61; Y; Y10; Y10S; Y10S530/00; Y10S530/808; Y10S530/82; Y10S530/825; Y10S930/00; Y10S930/01; Y10S930/12
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
40 words
A method for naturation of somatotropin protein which comprises contacting a dimethylsulfone or urea-dimethylsulfone solution containing said protein with a mild oxidizing agent for a time sufficient to form intramolecular disulfide bonds between cysteine residues contained in said somatotropin protein.
Claim 2
Independent Claim
40 words
A method for naturation of somatotropin protein which comprises contacting a dimethylsulfone or urea-dimethylsulfone solution containing said protein with a mild oxidizing agent for a time sufficient to form intramolecular disulfide bonds between cysteine residues contained in said somatotropin protein.
Claim 3
Dependent Claim
17 words
The method of claim 1 in which the somatotropin protein is natured in an aqueous dimethylsulfone solution.
Claim 4
Dependent Claim
20 words
The method of claim 2 in which the naturation is conducted in a dimethylsulfone concentration between about 0.5 and 2.4M.
Claim 5
Dependent Claim
16 words
The method of claim 3 in which the naturation is conducted at a pH above 9.
Claim 6
Dependent Claim
19 words
The method of claim 3 in which the naturation is conducted at a pH between about 9 and 12.
Claim 7
Dependent Claim
26 words
The method of claim 3 in which the naturation is conducted at a temperature above the freezing point of the solution and below about 30° C.
Claim 8
Dependent Claim
12 words
The method of claim 1 in which the somatotropin is bovine somatotropin.
Claim 9
Dependent Claim
43 words
The method of claim 1 in which the naturation is conducted at a dimethylsulfone concentration between about 0.5 and 2.4M; a temperature above the freezing point of the solution and below about 30° C.; and a pH between about 9 and about 12.
Claim 10
Dependent Claim
44 words
The method of claim 1 wherein the protein has been initially solubilized from refractile bodies of a host cell containing said protein by contacting said bodies with an effective amount and concentration of a weak denaturant at a pH effective to solubilize said protein.
Claim 11
Dependent Claim
21 words
The method of claim 9 in which the weak denaturant is selected from the group consisting of urea, dimethylsulfone and urea-dimethylsulfone.
Claim 12
Dependent Claim
26 words
The method of claim 10 in which the solubilization is conducted at a temperature above the freezing point of the solution and below about 25° C.
Claim 13
Dependent Claim
16 words
The method of claim 10 in which solubilization is conducted at a pH above about 9.
Claim 14
Dependent Claim
19 words
The method of claim 10 in which solubilization is conducted at a pH between about 9 and about 12.
Claim 15
Dependent Claim
26 words
The method of claim 10 in which solubilization is conducted at a pH between about 9 and about 12 and a temperature of about 4° C.
Claim 16
Dependent Claim
19 words
The method of claim 10 in which the host cell is a strain of the bacterial species Escherichia coli.
Claim 17
Dependent Claim
12 words
The method of claim 10 in which the somatotropin is bovine somatotropin.
Claim 18
Dependent Claim
19 words
The method of claim 10 in which the host cell is a strain of the bacterial species Escherichia coli.
Claim 19
Dependent Claim
48 words
The method of claim 17 in which said solution comprises urea and the urea concentration is between about 1.0M and 10M; the pH is maintained between about 9 and about 12; and the temperature is maintained above the freezing point of the solution and below about 25° C.
Claim 20
Dependent Claim
45 words
The method of claim 17 in which said solution comprises dimethylsulfone and the dimethylsulfone concentration is between about 0.5M and about 2.4M; the pH is maintained between about 11.25 and about 12; and the temperature is maintained between about 5° C. and about 30° C.
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) | 1990-09-14 | 1991-03-16 | 1991-09-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-09-14 | 1995-03-16 | 1995-09-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-09-14 | 1999-03-16 | 1999-09-15 | $7700.00 | Window Closed / Lapsed |