Purification of the HhaII restriction endonuclease from an overproducer Escherichia coli clone.

Journal Article

An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D-galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23-kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column.

Full Text

Duke Authors

Cited Authors

  • Kelly, S; Kaddurah-Daouk, R; Smith, HO

Published Date

  • December 5, 1985

Published In

Volume / Issue

  • 260 / 28

Start / End Page

  • 15339 - 15344

PubMed ID

  • 2999111

International Standard Serial Number (ISSN)

  • 0021-9258

Language

  • eng

Conference Location

  • United States