US4250262A
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US
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Method of preparing a purified glucosyltransferase
Assignee: ADA Forsyth Institute Inc
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Filing: 1979-12-14
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Granted: 1981-02-10
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Expires: 1999-12-14
Abstract
Abstract Immunization of animals with preparations containing more purified forms of glucosyltransferase (GTF) results in the presence of antibody in saliva demonstrable by functional inhibitions of enzyme activity and binding of radioactive enzyme. Serum antibody was also present. Immunized groups of animals had lower mean caries scores than comparably sham-immunized or nonimmunized control groups. Local immunization with GTF of serotype c or g of a Streptococcus mutans reduces the colonization, caries, and lesions caused by infection with S. mutans of serotype g (strain 6715) or c, or with serotype g or c, or with serotype a or g, respectively.
Inventors
- 👤 Martin A. Taubman
- 👤 Daniel J. Smith
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 01:17
CPC Classifications
A; A61; A61Q; A61Q11/00; A61K; A61K38/00; A61K38/16; A61K38/43; A61K38/45; A61K39/00; A61K39/02; A61K39/09; A61K39/092; A61K8/00; A61K8/18; A61K8/30; A61K8/64; A61K8/66; C; C12; C12N; C12N9/00; C12N9/10; C12N9/1048; C12N9/1051; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/8215; Y10S435/822; Y10S435/885
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
157 words
In a method of recovering glucosyltransferase (GTF) for use in immunization against dental caries, which method comprises: (a) culturing Streptococcus mutans in a medium containing glucose and essentially dialyzable nutrients to form a mixture of culture cells and supernatant; (b) removing the cells for the culture supernatant; (c) incubating the recovered supernatant with a polysaccharide to synthesize water-insoluble polysaccharide; (d) recovering the water-insoluble polysaccharide; (e) washing the polysaccharide to remove water-soluble contaminants; and (f) recovering the GTF from the water-insoluble polysaccharide, the improvement which comprises (i) incubating the washed water-insoluble polysaccharide from the supernatant with a denaturing solvent to break the bond between the water-insoluble polysaccharide and the GTF enzyme, (ii) separating the water-insoluble polysaccharide from the denaturing solvent containing the GTF enzyme, (iii) separating the denaturing solvent from the GTF in the supernatant, and (iv) concentrating the GTF enzyme to provide a concentrated GTF consisting of essentially purified GTF suitable for use for preventing dental caries.
Claim 2
Dependent Claim
157 words
In a method of recovering glucosyltransferase (GTF) for use in immunization against dental caries, which method comprises: (a) culturing Streptococcus mutans in a medium containing glucose and essentially dialyzable nutrients to form a mixture of culture cells and supernatant; (b) removing the cells for the culture supernatant; (c) incubating the recovered supernatant with a polysaccharide to synthesize water-insoluble polysaccharide; (d) recovering the water-insoluble polysaccharide; (e) washing the polysaccharide to remove water-soluble contaminants; and (f) recovering the GTF from the water-insoluble polysaccharide, the improvement which comprises (i) incubating the washed water-insoluble polysaccharide from the supernatant with a denaturing solvent to break the bond between the water-insoluble polysaccharide and the GTF enzyme, (ii) separating the water-insoluble polysaccharide from the denaturing solvent containing the GTF enzyme, (iii) separating the denaturing solvent from the GTF in the supernatant, and (iv) concentrating the GTF enzyme to provide a concentrated GTF consisting of essentially purified GTF suitable for use for preventing dental caries.
Claim 3
Dependent Claim
28 words
The method of claim 1 which comprises employing as the Streptococcus mutans a Streptococcus mutans selected from the group consisting of strain 6715, strain Ingbritt or strain E49.
Claim 4
Dependent Claim
31 words
The method of claim 1 wherein the culture medium is brain heart infusion containing about 1% glucose and which includes culturing the culture medium for about 20 hours at 37° C.
Claim 5
Dependent Claim
29 words
The method of claim 1 which includes incubating the culture supernatant with about 10% sucrose for about 48 hours at 37° C. and at a pH of about 6.5.
Claim 6
Dependent Claim
20 words
The method of claim 1 wherein the denaturing solvent is selected from the group consisting of quanidine-HCl, urea and dodecylsulfate.
Claim 7
Dependent Claim
31 words
The method of claim 1 which includes concentrating the GTF enzyme by gel filtration, and wherein the GTF is eluted on phenyl-sepharose in about 6 M quanidine-HCl as the denaturing solvent.
Claim 8
Dependent Claim
26 words
The method of claim 1 which includes employing a saline concentration of from about one to two times of the denaturing solvent with the water-insoluble polysaccharide.
Claim 9
Dependent Claim
13 words
The method of claim 1 which includes incubating the recovered supernatant with sucrose.
Claim 10
Dependent Claim
14 words
The method of claim 1 which includes concentrating the GTF enzyme by gel filtration.
Claim 11
Dependent Claim
23 words
The method of claim 1 wherein the denaturing solvent is guanidine-HCl and which includes removing the denaturing solvent from the supernatant by dialysis.
Claim 12
Dependent Claim
26 words
The method of claim 1 which includes concentrating a purified GTF enzyme to a specific activity of 2.7 and a GTF/FTF of greater than about 97%.
Claim 13
Dependent Claim
25 words
The method of claim 1 which includes preparing a vaccine for preventing dental caries wherein the concentrated GTF comprises the active ingredients of the vaccine.
Claim 14
Dependent Claim
178 words
In a method of recovering glucosyltransferase (GTF) for use in a vaccine for immunization against dental caries, which method comprises: (a) culturing Streptococcus mutans, selected from the group consisting of strain 6715, strain Ingbritt or strain E49, in a medium containing glucose and essentially dialyzable nutrients to form a mixture of culture cells and supernatant; (b) removing the cells for the culture supernatant; (c) incubating the recovered supernatant with sucrose to synthesize water-insoluble polysaccharide; (d) recovering the water-insoluble polysaccharide; (e) washing the polysaccharide to remove water-soluble contaminants; and (f) recovering the GTF from the water-insoluble polysaccharide, the improvement which comprises (i) incubating the washed water-insoluble polysaccharide from the supernatant with a guanidine-HCl denaturing solvent to break the bond between the water-insoluble polysaccharide and the GTF enzyme, (ii) separating the water-insoluble polysaccharide from the denaturing solvent containing the GTF enzyme, (iii) separating the denaturing solvent from the GTF in the supernatant, and (iv) concentrating the GTF enzyme by gel filtration to provide an eluent consisting of essentially purified GTF suitable for use in vaccine form for preventing dental caries.
Claim 15
Dependent Claim
178 words
In a method of recovering glucosyltransferase (GTF) for use in a vaccine for immunization against dental caries, which method comprises: (a) culturing Streptococcus mutans, selected from the group consisting of strain 6715, strain Ingbritt or strain E49, in a medium containing glucose and essentially dialyzable nutrients to form a mixture of culture cells and supernatant; (b) removing the cells for the culture supernatant; (c) incubating the recovered supernatant with sucrose to synthesize water-insoluble polysaccharide; (d) recovering the water-insoluble polysaccharide; (e) washing the polysaccharide to remove water-soluble contaminants; and (f) recovering the GTF from the water-insoluble polysaccharide, the improvement which comprises (i) incubating the washed water-insoluble polysaccharide from the supernatant with a guanidine-HCl denaturing solvent to break the bond between the water-insoluble polysaccharide and the GTF enzyme, (ii) separating the water-insoluble polysaccharide from the denaturing solvent containing the GTF enzyme, (iii) separating the denaturing solvent from the GTF in the supernatant, and (iv) concentrating the GTF enzyme by gel filtration to provide an eluent consisting of essentially purified GTF suitable for use in vaccine form for preventing dental caries.
References Cited (Backward Citations)
US3993747A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-02-10 | 1984-08-11 | 1985-02-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-02-10 | 1988-08-11 | 1989-02-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-02-10 | 1992-08-11 | 1993-02-10 | $7700.00 | Window Closed / Lapsed |