ABSTRACT
The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.
Subject(s)
Chromatography, Affinity/methods , DNA-Binding Proteins/isolation & purification , Animals , Base Sequence , DNA/chemistry , DNA Primers , DNA-Binding Proteins/chemistry , DNA-Directed DNA Polymerase/isolation & purification , DNA-Directed RNA Polymerases/isolation & purification , Telomerase/isolation & purification , Templates, Genetic , Transcription Factors/isolation & purificationABSTRACT
Syntrophins have been proposed to serve as adapter proteins. Syntrophins are found in the dystrophin glycoprotein complex (DGC); defects in the constituents of this complex are linked to various muscular dystrophies. Blot overlay experiments demonstrate that alpha-dystroglycan, beta-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse alpha1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to also bind Grb2 in a Ca2+-independent manner. This binding was localized to the proline rich sequences adjacent to and overlapping with the N-terminal pleckstrin homology domain (PH1). Grb2 bound syntrophin with an apparent KD of 563 +/- 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity, does not bind these syntrophin sequences.
Subject(s)
Adaptor Proteins, Signal Transducing , Membrane Proteins/metabolism , Muscle Proteins/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Blood Proteins/metabolism , Calcium-Binding Proteins , Cytoskeletal Proteins , Dystroglycans , GRB2 Adaptor Protein , Membrane Glycoproteins , Mice , Molecular Sequence Data , Peptide Fragments/metabolism , Phosphoproteins/metabolism , Protein Binding , Rabbits , Recombinant Fusion Proteins/metabolism , Signal Transduction , src Homology DomainsABSTRACT
Syntrophins are known to self-associate to form oligomers. Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and were found to also oligomerize and in a micromolar Ca(2+)-dependent manner. The oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to self-associate, and calmodulin or Ca(2+)-chelating agents such as ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Since calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca(2+), Ca(2+) binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.
