Method for producing the Eag I restriction endonuclease and methylase
First Claim
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1. Isolated DNA coding for the EagI restriction endonuclease, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
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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.
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10 Claims
- 1. Isolated DNA coding for the EagI restriction endonuclease, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
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2. 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.
- 3. Isolated DNA coding for the EagI restriction endonuclease and methylase, wherein the isolated DNA is obtainable from the vector pEagRM2.9.
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9. A method of cloning DNA coding for an EagI restriction endonuclease comprising:
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(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.
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10. A method of producing EagI restriction endonuclease comprising:
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(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.
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Specification