Glutathione S-transferase fusion proteins as an affinity reagent for rapid isolation of specific sequence directly from genomic DNA.
We describe a DNA binding assay for isolation of specific sequence(s) recognized by protein of interest directly from genomic or cosmid DNA. In our assay, the protein is fused to the glutathione-S-transferase and bound to glutathione-Sepharose beads. Then the immobilized fusion protein can be used to search for DNA fragment(s) that interact specifically with the protein of interest. As an example of such an approach, we identified and cloned a few prokaryotic oriC regions using the initiator DnaA protein fused to the glutathione-S-transferase.
Majka
J
J
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 12, 53-114 Wroclaw, Poland.
Jakimowicz
D
D
Zarko-Postawka
M
M
Zakrzewska-Czerwinska
J
J
D001426:Bacterial Proteins / Q000096:biosynthesis; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016680:Genome, Bacterial; D005982:Glutathione Transferase / Q000096:biosynthesis; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018741:Replication Origin; D013302:Streptomyces / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase