Detection, purification, and characterization of cDNA clones encoding DNA-binding proteins.
In this unit, an appropriate recombinant clone is detected in an expression library with a DNA recognition-site probe, purified, and shown to encode a DNA-binding domain of defined sequence specificity. The strategy described below obviates purification of a sequence-specific DNA-binding protein for the purpose of isolating its gene; it simply requires a suitable cDNA library constructed in the expression vector lgt11 and a DNA recognition-site probe. The basic protocol enables the detection and purification of clones encoding sequence-specific DNA-binding proteins. The alternate protocol describes a denaturation/renaturation procedure that can increase detection of certain clones. The support protocol provides a rapid means of characterizing the DNA-binding activities of the proteins encoded by the cloned cDNAs.
Singh
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University of Chicago, Chicago, Illinois, USA.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D015342:DNA Probes; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D019791:Guanidine; D006801:Humans; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015342:DNA Probes; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019791:Guanidine