Ni Affinity Column Research Articles

RAR antagonists diminish the level of DNA binding by the RAR/RXR heterodimer.

A purified RAR/RXR-DeltaAB heterodimer was obtained by production of His-tagged RAR and untagged RXR in Escherichia coli, followed by combined purification on a Ni(2+) affinity column using excess RXR extract, and finally a gel filtration chromatography step to isolate a pure heterodimer. The purified heterodimer preparation bound 9-cisRA at a level of 0.85-0.95 mol of binding sites per mole of protein monomer. Titration of a 26 kDa fluorescent labeled fragment of the SRC-1 coactivator protein with the purified heterodimer in the presence of the agonist 9-cisRA yielded a binding affinity near 300 nM, whereas no binding was observed in the absence of agonist. Binding of the purified heterodimer to a DR5 target was identical in the absence of ligand and in the presence of 9-cisRA. Competition by unlabeled specific and nonspecific DNA allowed us to demonstrate that the binding curve was bimodal. The first phase of binding was highly specific and of high affinity. This phase also exhibited a high degree of cooperativity in the binding profile. Nonspecific DNA efficiently competes for the second phase. Thus, the first phase of binding likely corresponds to the formation of the specific heterodimer complex in which heterodimerization is energetically coupled to DNA binding. While agonist binding had no effect on the apparent affinity of the heterodimer for DR5, a series of antagonists significantly destabilized the heterodimer-DR5 complex, either through a direct decrease in the affinity of the protein for the DNA or through destabilization of the heterodimer itself. Impeding the interaction between the heterodimer and DNA appears as an additional mechanism of antagonist action of varying efficiency, depending upon the chemical structure of the antagonist.

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli.

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.

Expression and rapid one-step purification of biologically active His-tagged factor C by Ni(2+) affinity column chromatography.

Factor C is an unusual extracellular protein capable of inducing cytodifferentiation in certain Streptomyces strains. The protein is produced by Streptomyces griseus 45H at such a low amount that the study of its mode of action was hindered by the shortage of purified protein. We report here the expression of C-terminally hexa-His-tagged factor C in Streptomyces lividans and Escherichia coli. Expression in S. lividans is low while in E. coli it is relatively high, yielding about 5--10 mg of biologically fully active protein per liter culture.

Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme.

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni(2+) affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.

A simple purification and fluorescent assay method of the poliovirus 3C protease searching for specific inhibitors

Picornaviruses such as poliovirus, foot-and-mouth disease virus, and encephalomyocarditis virus produce their proteins by translating their genomic RNA, injected within the host cell, into a precursor polyprotein, which is then subjected to precise processing. The polyprotein is cleaved into mature proteins predominantly by the viral 3C protease. A simple purification and assay method for poliovirus 3C protease for use for screening for inhibitors of the 3C protease is described. A poliovirus cDNA fragment containing the 3C protease coding region was inserted into pET22b vector and expressed in Escherichia coli. The His-tagged protein (3CD′-His) was purified by a Ni-affinity column and the activity of the purified enzyme was measured by a fluorescent assay with a fluorogenic substrate containing the 3C-specific cleavage site, MocAc-MEALF QG PLQY-Dnp. The kinetic parameters calculated from the Lineweaver–Burk plot and the effects of inhibitors showed that E. coli expression with His tag and the assay using the fluorogenic substrate are efficient, simple and sensitive methods for purifying the 3C protease, and measuring its activity.

Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins.

The nda1+ and nda4+ genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, respectively, which are required for the early stages of DNA replication. The budding yeast Mcm proteins display cell-cycle dependent localization. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be components of a replication licensing factor, a positive factor for the onset of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kDa and 80 kDa proteins, respectively. Their immunolocalization was examined in wild-type cells and in various cell-cycle mutants. Both Nda1 and Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in G1, S, and G2 phases, these proteins were also enriched in the nucleus. These results indicate that the dramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 proteins to be executed. The histidine-tagged nda1+ gene was constructed and integrated into the chromosome to replace the wild-type nda1+ gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-affinity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.