Immobilization of the restriction endonuclease PstI |
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Authors: | Pratap Singh |
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Institution: | (1) Southern Biotech, Inc., 128 S. Moon Avenue, 33511 Brandon, FL;(2) Present address: Clinical Chemistry Division, American Dade, PO Box 520276, 33152 Miami, Florida |
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Abstract: | PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed
a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide
in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity.
It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation
of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity
of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible
for inactive enzyme preparations. |
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Keywords: | Restriction endonuclease immobilization endonuclease PstI immobilization of PstI immobilization of the endonuclease immobilization of restriction endonuclease PstI agarose enzyme immobilization structure-function relationship of endonuclease DNA cleavage by immobilized enzyme polyacrylamide enzyme immobilization on endonuclease presence of active amino groups on immobilized enzyme treatment with crosslinking agent |
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