Cloning of Sequence-Specific DNA-Binding Proteins by Screening λ cDNA Expression Libraries with Radiolabelled Binding-Site Probes

摘要: The isolation of DNA-binding, transcription factors has been facilitated by a novel strategy that depends on the functional expression in E. coli high levels DNA-binding domain (DBD) factor studied. This procedure (Singh et al., 1988, Vinson 1988) , which is essentially modification original antibody screening λ gtll libraries (Young & Davis 1983) can be outlined as follows (see Fig. 1) : A cDNA library constructed an inducible prokaryotic vector such bacteriophage vectors gt11 or ZAPII. Both produce proteins fused with N-terminal portion s-galactosidase, and each case synthesis fusion protein under control lac repressor. After plating library, phage plaques are blotted onto filters had previously soaked solution IPTG, lactose analogue. IPTG inactivates repressor, thus induces situ from recombinants. presence desired then detected incubating radiolabelled DNA probe containing binding site for factor, conditions comparable to those used footprint gel-retardation experiments. washing autoradiography filters, phages deemed positive eluted replated. Parallel prepared, induced probed either genuine binding-site probe, or, ideally point-mutated version it known abolish vitro (Fig. 1).