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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Singh, P. Immobilization of the restriction endonuclease PstI. Appl Biochem Biotechnol 9, 161–172 (1984). https://doi.org/10.1007/BF02798750
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DOI: https://doi.org/10.1007/BF02798750
Index Entries
- 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