EP0192629A2
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EP
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Method of somatotropin solubilization and naturation
Assignee: Monsanto Technology LLC
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Filing: 1986-02-21
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Granted: —
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Expires: 2006-02-21
Abstract
Abstract e 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 urea or dimethylsulfone solution can be effectively used to solubilize refractile bodies containing such somatotropin protein. Once solubilized, somatotropin protein can be natured in a urea or dimethylsulfone solution 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 Andrew Bentle
- 👤 James William Mitchell
- 👤 Stephen Bradley Storrs
- 👤 Grant Tsuyoshi Shimamoto
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 10:47
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/96; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K14/00; C07K14/435; C07K14/575; C07K14/61
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
76 words
A method for solubilization and naturation 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 an aqueous urea or dimethylsulfone solution at a pH effective to accomplish solubilization of said protein and then contacting the solution containing said protein with a mild oxidizing agent for a time sufficient to form intramolecular disulfide bonds between cysteine residues contained in said protein.
Claim 2
Independent Claim
76 words
A method for solubilization and naturation 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 an aqueous urea or dimethylsulfone solution at a pH effective to accomplish solubilization of said protein and then contacting the solution containing said protein with a mild oxidizing agent for a time sufficient to form intramolecular disulfide bonds between cysteine residues contained in said protein.
Claim 3
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 urea or dimethylsulfone at a pH effective to accomplish solubilization of said protein.
Claim 4
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 urea or dimethylsulfone at a pH effective to accomplish solubilization of said protein.
Claim 5
Dependent Claim
17 words
The method of Claim 2 in which the refractile bodies are solubilized in an aqueous urea solution.
Claim 6
Dependent Claim
25 words
The method of Claim 3 in which the solubilization is conducted at a temperature above the freezing point of the solution and below about 25°C.
Claim 7
Dependent Claim
16 words
The method of Claim 3 in which solubilization is conducted at a pH above about 9.
Claim 8
Dependent Claim
18 words
The method of Claim 3 in which solubilization is conducted at a pH between about 9 and 12.
Claim 9
Dependent Claim
24 words
The method of Claim 3 in which solubilization is conducted at a pH between about 9 and 12 and a temperature of about 4°C.
Claim 10
Dependent Claim
18 words
The method of Claim 3 in which the host cell is a strain of the bacterial species Escherichiacoli.
Claim 11
Dependent Claim
16 words
The method of Claim 3 in which solubilization is conducted in the absence of reducing agent.
Claim 12
Dependent Claim
16 words
The method of Claim 3 in which solubilization is conducted in the presence of reducing agent.
Claim 13
Dependent Claim
14 words
The method of Claim 3 in which the somatotropin is bovine or porcine somatotropin.
Claim 14
Dependent Claim
21 words
The method of Claim 11 in which the host cell is a strain of the bacterial species Esche h chia coli.
Claim 15
Dependent Claim
44 words
The method of Claim 12 in which the urea concentration is between about 1 .0 M and 10 M; the pH is maintained between about 9 and 12; and the temperature is maintained above the freezing point of the solution and below about 25°C.
Claim 16
Dependent Claim
20 words
The method of Claim 13 in which the pH is maintained in said range using an effective amount of Tris-(methylhydroxy)aminomethane.
Claim 17
Independent Claim
40 words
A method for naturation of somatotropin protein which comprises contacting a urea or 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 18
Independent Claim
40 words
A method for naturation of somatotropin protein which comprises contacting a urea or 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 19
Dependent Claim
17 words
The method of Claim 15 in which the somatotropin protein is natured in an aqueous urea solution.
Claim 20
Dependent Claim
21 words
The method of Claim 16 in which the naturation is conducted in a urea concentration between about 3 and 5 M.
Claim 21
Dependent Claim
16 words
The method of Claim 16 in which the naturation is conducted at a pH above 9.
Claim 22
Dependent Claim
19 words
The method of Claim 16 in which the naturation is conducted at a pH between about 9 and 12.
Claim 23
Dependent Claim
25 words
The method of Claim 16 in which the naturation is conducted at a temperature above the freezing point of the solution and below about 25°C.
Claim 24
Dependent Claim
17 words
The method of Claim 16 in which the naturation is conducted in the presence of reducing agent.
Claim 25
Dependent Claim
17 words
The method of Claim 16 in which the naturation is conducted in the absence of reducing agent.
Claim 26
Dependent Claim
14 words
The method of Claim 11 in which the somatotropin is bovine or porcine somatotropin.
Claim 27
Dependent Claim
42 words
The method of Claim 23 in which the naturation is conducted at a urea concentration between about 2 and 5 M; a temperature above the freezing point of the solution and below about 25°C; and a pH between about 9 and 12.
Claim 28
Dependent Claim
17 words
The method of Claim 2 in which the refractile bodies are solubilized in an aqueous dimethylsulfone solution.
Claim 29
Dependent Claim
17 words
The method of Claim 15 in which the somatotropin protein is natured in an aqueous dimethylsulfone solution.
References Cited (Backward Citations)
No citation records indexed.
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