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      Permanent link:
      https://www.ias.ac.in/article/fulltext/jbsc/024/03/0269-0277

    • Keywords

       

      Polymerase chain reaction; molecular cloning; endonuclease; methyltransferase; gel retardation assay

    • Abstract

       

      The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.

    • Author Affiliations

       

      Siddamadappa Chandrashekaran1 2 Padmanabhan Babu2 Valakunja Nagaraja1

      1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560 012, India
      2. Industrial Suburb, Peenya, Bangalore Genei Pvt. Ltd, Bangalore - 560 058, India
    • Dates

       
  • Journal of Biosciences | News

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