Purification Of Sequence-specific DNA-binding Proteins Research Articles

Detection and Purification of Sequence-Specific DNA Binding Protein

A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with32P-labeled DNA probe in buffer in the presence of excess of nonspecificE. coliDNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of theDrosophilaclone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA–Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.

36] Magnetic DNA affinity purification of yeast transcription factor

This chapter describes the preparation of magnetic DNA affinity beads and their use in the purification of sequence-specific DNA-binding proteins. The protein chosen for the illustration of the method is yeast transcription factor z––one of the most complex transcription factors characterized so far. The chapter presents an improved variant of the previously reported magnetic DNA affinity purification method: starting with a partially purified protein fraction (purity < 0.2%), essentially the pure preparations of yeast transcription factor z were obtained in less than 30 min with a single adsorption step. Improved purity and yields were obtained by saturating the DNA affinity beads with the specific DNA binding protein and by including competitor DNA only during the washing of the beads. With the same starting material, this 30-min procedure yielded higher purity than the normal method, which uses columns and requires several days.

A high-capacity column for affinity purification of sequence-specific DNA-binding proteins.

A high-capacity column for affinity purification of sequence-specific DNA-binding proteins.

Purification of DNA-binding transcription factors by their selective adsorption on the affinity latex particles.

A simple method with the use of affinity latex particles has been developed for the fast and efficient purification of sequence-specific DNA-binding proteins on the basis of their ability to selectively bind to their target sequences. Complementary oligodeoxynucleotides that contained a recognition site for a sequence-specific DNA-binding protein were chemically synthesized, annealed and ligated to give oligomers. The oligomers were coupled to latex particles, composed of polyglycidyl methacrylate, using cyanogen bromide to yield affinity latex particles. The concentration of covalently bound DNA on the affinity latex particles was 6 times as much DNA per ml as that in the Sepharose resin conventionally used. By sequential batch-wise procedures with the affinity particles, one of the sequence-specific DNA binding transcription factors, ATF or E4TF3, was quickly and efficiently purified to homogeneity from either a protein fraction in which the factor was enriched or a crude cell extract.

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI) — Generation of protein sequence information

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.