Patent US4996151A successfully refreshed from Google Patents.
US4996151A
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Method for producing the Eag I restriction endonuclease and methylase
Assignee: New England Biolabs Inc
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Filing: 1988-05-19
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Granted: 1991-02-26
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Expires: 2008-05-19
Abstract
Abstract The present invention is directed to a method for cloning and producing the Eag I restriction endonuclease by (1) introducing the restriction endonuclease gene from E. agglomerans ATCC into a host whereby the restriction gene is expressed; (2) fermenting the host which contains the vector encoding and expressing the Eag I restriction endonuclease, and (3) purifying the Eag I restriction endonuclease from the fermented host which contains the vector encoding and expressing the Eag I restriction endonuclease activity.
Inventors
- 👤 Joan E. Brooks
- 👤 Laura A. Sznyter
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 14:36
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/16; C12N9/22; C12N9/10; C12N9/1003; C12N9/1007
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
18 words
Isolated DNA coding for the EagI restriction endonuclease, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
Claim 2
Dependent Claim
18 words
Isolated DNA coding for the EagI restriction endonuclease, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
Claim 3
Dependent Claim
27 words
A recombinant DNA vector comprising a vector into which a DNA segment coding for the EagI endonuclease produced from E. agglomerans ATCC No. 53769 has been inserted.
Claim 4
Dependent Claim
27 words
A recombinant DNA vector comprising a vector into which a DNA segment coding for the EagI endonuclease produced from E. agglomerans ATCC No. 53769 has been inserted.
Claim 5
Dependent Claim
20 words
Isolated DNA coding for the EagI restriction endonuclease and methylase, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
Claim 6
Dependent Claim
20 words
Isolated DNA coding for the EagI restriction endonuclease and methylase, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
Claim 7
Dependent Claim
11 words
A cloning vector which comprises the isolated DNA of claim 1.
Claim 8
Dependent Claim
11 words
A cloning vector which comprises the isolated DNA of claim 3.
Claim 9
Dependent Claim
12 words
The cloning vector of claim 5, wherein the cloning vector comprises pEagRM2.9.
Claim 10
Dependent Claim
12 words
A host cell transformed by the vector of claim 5 or 6.
Claim 11
Dependent Claim
31 words
A method of producing EagI restriction endonuclease comprising culturing a host cell transformed with the vector of claim 4, 5 or 6 under conditions suitable for the expression of said endonuclease.
Claim 12
Independent Claim
155 words
A method of cloning DNA coding for an EagI restriction endonuclease comprising: (a) purifying DNA from E. agglomerans ATCC No. 53769; (b) digesting the purified DNA with PstI to form DNA fragments; (c) ligating the DNA fragments into cloning vector pLSN3; (d) transforming a host cell with the cloning vector of step (c) to form a cell library; (e) purifying recombinant vectors from the cell library to form a plasmid library; (f) contacting the plasmid library of step (e) with NotI to form a digestion pool, transforming the digestion pool into a host cell, and screening for the presence of one or more cloning vectors containing DNA coding for an EagI methylase; (g) screening the cloning vectors of step (f) which contain DNA coding for EagI methylase for the presence of DNA coding for an EagI restriction endonuclease; and (h) isolating the cloning vectors of step (g) which contain DNA coding for EagI restriction endonuclease.
Claim 13
Independent Claim
155 words
A method of cloning DNA coding for an EagI restriction endonuclease comprising: (a) purifying DNA from E. agglomerans ATCC No. 53769; (b) digesting the purified DNA with PstI to form DNA fragments; (c) ligating the DNA fragments into cloning vector pLSN3; (d) transforming a host cell with the cloning vector of step (c) to form a cell library; (e) purifying recombinant vectors from the cell library to form a plasmid library; (f) contacting the plasmid library of step (e) with NotI to form a digestion pool, transforming the digestion pool into a host cell, and screening for the presence of one or more cloning vectors containing DNA coding for an EagI methylase; (g) screening the cloning vectors of step (f) which contain DNA coding for EagI methylase for the presence of DNA coding for an EagI restriction endonuclease; and (h) isolating the cloning vectors of step (g) which contain DNA coding for EagI restriction endonuclease.
Claim 14
Independent Claim
173 words
A method of producing EagI restriction endonuclease comprising: (a) purifying DNA from E. agglomerans ATCC No. 53769; (b) digesting the purified DNA with PstI to form DNA fragments; (c) ligating the DNA fragments into cloning vector pLSN3; (d) transforming a host cell with the cloning vector of step (c) to form a cell library; (e) purifying recombinant vectors from the cell library to form a plasmid library; (f) contacting the plasmid library of step (e) with NotI to form a digestion pool, transforming the digestion pool into a host cell, and screening for the presence of one or more cloning vectors containing DNA coding for an EagI methylase; (g) screening the cloning vectors of step (F) which contain DNA coding for EagI methylase for the presence of DNA coding for an EagI restriction endonuclease; (h) isolating the cloning vectors of step (g) which contain DNA coding for EagI restriction endonuclease; and (i) culturing a host cell transformed with the cloning vector of step (h) under conditions suitable for expression of EagI restriction endonuclease.
Claim 15
Independent Claim
173 words
A method of producing EagI restriction endonuclease comprising: (a) purifying DNA from E. agglomerans ATCC No. 53769; (b) digesting the purified DNA with PstI to form DNA fragments; (c) ligating the DNA fragments into cloning vector pLSN3; (d) transforming a host cell with the cloning vector of step (c) to form a cell library; (e) purifying recombinant vectors from the cell library to form a plasmid library; (f) contacting the plasmid library of step (e) with NotI to form a digestion pool, transforming the digestion pool into a host cell, and screening for the presence of one or more cloning vectors containing DNA coding for an EagI methylase; (g) screening the cloning vectors of step (F) which contain DNA coding for EagI methylase for the presence of DNA coding for an EagI restriction endonuclease; (h) isolating the cloning vectors of step (g) which contain DNA coding for EagI restriction endonuclease; and (i) culturing a host cell transformed with the cloning vector of step (h) under conditions suitable for expression of EagI restriction endonuclease.
References Cited (Backward Citations)
EP0193413A2
Cited
US6707079B2
Cited
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