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1.
Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.  相似文献   

2.
J Chou  B Roizman 《Journal of virology》1989,63(3):1059-1068
The terminal 500-base-pair alpha sequence of the herpes simplex virus 1 genome contains signals for cleavage (Pac1 and Pac2) of unit-length DNA molecules from concatemers in unique stretches of sequences designated Ub and Uc, respectively, and a cis site for cleavage designated DR1. We report that nuclear extracts from infected cells contain factors which form two DNA-virus-specific protein complexes with components of the a sequence. Purification of the factors forming the V2 complex yielded a protein with an apparent molecular weight of 82,000 binding to DNA in a non-sequence-specific manner. Addition of Mg2+ to the purified protein-DNA probe mixture resulted in exonucleolytic degradation of the DNA. The protein was identified as the virus-specific DNase with monoclonal antibody specific for the viral enzyme. The purification of the proteins forming the V4 complex yielded two proteins with molecular weights of greater than 250,000 and 140,000 corresponding to infected cell protein 1 and to an as yet unidentified protein, respectively. These proteins formed two DNA sequence-common bands with a number of DNA probes and one sequence-specific band with probes containing both Pac2 and DR1 but not with probes containing either site alone or Pac1 and DR1. Since the DNA probe containing Pac2 and DR1 inserted into viral genome or into amplicons induced specific cleavage of the DR1 sequence whereas the nonreactive probes failed to induce the cleavage, the formation of this sequence-specific DNA-protein complex is significant and may reflect a DNA-protein interaction essential for cleavage. The possible role of the proteins identified in this study for the cleavage-packaging of viral DNA into capsids is presented.  相似文献   

3.
The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure. These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein. From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein. We note, however, that their conformation is not unique and can be modulated by cAMP. The two altered proteins, CAP and CAPc, bind to the lactose promoter, giving rise to specific DNA-protein complexes in the absence of cAMP and promote initiation of specific lac messenger RNA synthesis.  相似文献   

4.
Fast gel electrophoresis to analyze DNA-protein interactions   总被引:1,自引:0,他引:1  
A rapid method for electrophoresis of DNA-protein complexes is described. Popular "gel-shift" assays are performed using Pharmacia PhastSystem with its convenience of pre-cast gels and buffer strips and microprocessor-controlled high-resolution separation. Using this system, the products of a DNA binding reaction (DNA-protein complexes) can be separated from "free" DNA in less than one hour. DNA fragments as well as oligonucleotides have been used as targets with partially purified extracts containing sequence-specific DNA binding proteins. We present here a comparison of results of gel-shift assays obtained by conventional techniques and by our rapid "PhastShift" method which reduces the time, effort and technical expertise necessary to obtain reproducible results.  相似文献   

5.
6.
Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.  相似文献   

7.
DNA- and RNA-binding proteins of chromatin from Escherichia coli   总被引:3,自引:0,他引:3  
The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sj?stad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.  相似文献   

8.
Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Each step contributes to the product cost and product loss. In this research, a cyclic process integrating commercially available ultrafiltration membranes and chromatographic resin beads was developed to achieve the same goal in one device. The device consisted of ion exchange beads on the shell side of a hollow fiber ultrafiltration module. Loading of proteins on the stationary phase on the shell side was carried out for a period of 5-20 min from the permeate on the shell side produced from tube-side feed in ultrafiltration. The eluent was then introduced either from the shell-side inlet or tube-side inlet; the chromatographic fractions were collected from the shell-side outlet. The column was regenerated/washed next to start a new cycle. Systems studied in this cyclic process include the following binary mixtures: myoglobin and beta-lactoglobulin; hemoglobin and bovine serum albumin; and myoglobin and alpha-lactalbumin. Excellent resolutions of the proteins were obtained. A yeast-based cellular suspension containing a mixture of myoglobin and alpha-lactalbumin was also applied to this device. The target proteins were recovered and purified successfully. The cyclic process-based device integrates clarification, concentration, and chromatographic purification of biomolecules and is suitable for both extracellular and intracellular products.  相似文献   

9.
A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.  相似文献   

10.
The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.  相似文献   

11.
Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn2+ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.  相似文献   

12.
Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.  相似文献   

13.
A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.  相似文献   

14.
Improved purification of rat intestinal lactase   总被引:2,自引:0,他引:2  
A rapid and improved method to obtain purified lactase from rat intestine is described. The purification procedure involved only two chromatographic steps. The degree of purification was far above (500 fold) the values reached with classical methods. Rabbit antisera raised to the purified lactase were characterized using conventional immunological techniques. The specificity of the lactase antibodies was confirmed by the lack of interference on maltase, aminopeptidase and alkaline phosphatase activities measured after papain extraction of the membrane proteins.  相似文献   

15.
The nucleoprotein structure of telomeres from Euplotes crassus was studied by using nuclease and chemical footprinting. The macronuclear telomeres were found to exist as DNA-protein complexes that are resistant to micrococcal nuclease digestion. Each complex encompassed 85 to 130 base pairs of macronuclear DNA and appeared to consist of two structural domains that are characterized by dissimilar DNA-protein interactions. Dimethyl sulfate footprinting demonstrated that very sequence-specific and salt-stable interactions occur in the most terminal region of each complex. DNase I footprinting indicated that DNA in the region 30 to 120 base-pairs from the 5' end lies on a protein surface; the interactions in this region of the complex are unlikely to be sequence specific. A 50-kilodalton telomere-binding protein was isolated. Binding of this protein protected telomeric DNA from BAL 31 digestion and gave rise to many of the sequence-specific DNA-protein interactions that were observed in vivo. The telomeric complexes from E. crassus were very similar in overall structure to the complexes found at Oxytricha telomeres. However, telomeric complexes from the two ciliates showed significant differences in internal organization. The telomeric DNA, the telomere-binding proteins, and the resultant DNA-protein interactions were all somewhat different. The telomere-binding proteins from the two ciliates were found to be less closely conserved than might have been expected. It appears that the proteins are tailored to match their cognate telomeric DNA.  相似文献   

16.
Oncogene protein products from avian myeloblastosis virus, p48v-myb, and from avian leukemia virus E26, p135gag-myb-ets, are located predominantly in the nucleus of nonproducer bone marrow cell clones, as revealed by indirect immunofluorescence. Both oncogene proteins were purified by immunoaffinity chromatography using monoclonal antibodies against p19 and immunoglobulins specific for myb, which was expressed in bacteria for antibody production. The purified proteins bind to DNA in vitro. In contrast, purified p135gag-myb-ets proteins from several mutants of E26 virus, temperature-sensitive for myeloblast transformation, either lost their abilities to bind to DNA or exhibited highly thermolabile DNA-protein interactions in vitro. DNA binding of AMV and E26 oncogene proteins is inhibited by myb-specific immunoglobulins. Our results suggest that lesions in the myb oncogene affect transformation as well as DNA binding of myb proteins in vitro.  相似文献   

17.
Aminopropylcobalamin (AP-Cbl), prepared from 3-chloropropylamine and cob(I)alamin, was immobilized on CNBr-activated Sephacryl beads. The product, Sephacryl-aminopropylcobalamin, contained ca. 1 μmol of AP-Cbl/ml of beads. Cobalamin-binding proteins in biological fluids were adsorbed selectively and quantitatively by Sephacryl-aminopropylcobalamin. After being washed to remove extraneous protein, the beads were photoirradiated to release the cobalamin-binding proteins as their aquacobalamin complexes. The latter could be converted to labeled cyanocobalamin complexes by treatment with [14C]KCN. The efficacy of this affinity chromatographic method is illustrated by the purification to near homogeneity and in high yield of three representative proteins: transcobalamin II from rabbit serum, intrinsic factor from human gastric juice, and R binder from human saliva.  相似文献   

18.
Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.  相似文献   

19.
Ribosomal proteins (r-proteins) constitute a considerable part of the cell proteome. Although their primary role in the cell is to serve as integral components of protein synthesis machinery, ribosomes, many of them have functions beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with other cell components. Extraribosomal activities of some ribosomal proteins were observed as early as the 1970s–1980s. In recent years, both the list of moonlighting r-proteins and the repertoire of their additional functions beyond the ribosome was greatly expanded, mainly owing to new techniques developed for dissecting RNA/DNA-protein or protein-protein interactions within functional complexes involved in various cell processes. The review surveys information on the extraribosomal functions demonstrated experimentally or presumed for bacterial r-proteins.  相似文献   

20.
Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.  相似文献   

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