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BMC Biochemistry Feb 2013The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers...
BACKGROUND
The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro.
RESULTS
We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies.
CONCLUSIONS
Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.
Topics: Cell Cycle Proteins; DNA-Binding Proteins; Escherichia coli; Gene Expression Regulation, Fungal; Minichromosome Maintenance Complex Component 4; Minichromosome Maintenance Complex Component 6; Minichromosome Maintenance Complex Component 7; Protein Binding; Protein Multimerization; Protein Subunits; Recombinant Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 23444842
DOI: 10.1186/1471-2091-14-5 -
Acta Crystallographica. Section F,... Sep 2010The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires...
The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (DeltaN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 A resolution. Swi5-DeltaN180Sfr1 was crystallized in space group P2(1)2(1)2 and the crystals diffracted to 2.3 A resolution. The crystals of Swi5 and Swi5-DeltaN180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.
Topics: Crystallization; Gene Expression; Protein Binding; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 20823543
DOI: 10.1107/S1744309110032239 -
Molecular and Cellular Biology Jun 1990Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4... (Comparative Study)
Comparative Study
Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.
Topics: Amino Acid Sequence; Base Sequence; Chromatography, Affinity; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Gene Expression; Kinetics; Molecular Sequence Data; Molecular Weight; Oligonucleotide Probes; Protein Binding; Protein Conformation; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Nucleic Acid; Software; Transcription Factors
PubMed: 2188103
DOI: 10.1128/mcb.10.6.2916-2923.1990 -
Protein Expression and Purification Jan 2018Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation....
Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.
Topics: Animals; Circular Dichroism; Cobalt; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Histone Deacetylases; Humans; Recombinant Proteins; Repressor Proteins; Sf9 Cells
PubMed: 28843507
DOI: 10.1016/j.pep.2017.08.009 -
Journal of Lipid Research Sep 1982Lipid transfer activities from human plasma have been characterized to determine whether triglyceride and cholesteryl ester transfer proteins are identical. After...
Lipid transfer activities from human plasma have been characterized to determine whether triglyceride and cholesteryl ester transfer proteins are identical. After sequential purification by phenyl-Sepharose, CM-cellulose, chromatofocusing, and gel filtration, both triglyceride and cholesteryl ester transfer activities were purified approximately 15,000-fold compared to lipoprotein-deficient plasma, with a 14% recovery of both transfer activities. The gel filtration fraction showed two bands, Mr 58,300 and 66,400, as determined by electrophoresis in sodium dodecyl sulfate. Two samples, each containing predominately one of the two bands, were obtained by selectively combining the eluates from the gel filtration column. The specific activities of triglyceride and cholesteryl ester transfer promoted by the larger protein were within 10% of those for the smaller protein. The relative rates of transfer for cholesteryl ester, triglyceride, retinyl ester, and cholesteryl ether for each fraction were the same. The transfer of triglyceride by either the large or small molecular weight component was almost completely inhibited by mercurial compounds, whereas cholesteryl ester transfer was relatively unaffected. We conclude that triglyceride and cholesteryl ester are transferred by the same plasma protein(s).
Topics: Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Isoelectric Focusing; Lipoproteins; Lipoproteins, LDL; Liposomes
PubMed: 7142814
DOI: No ID Found -
Protein Science : a Publication of the... Apr 2019The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of...
The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.
Topics: Binding Sites; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Immobilized Proteins; Recombinant Proteins
PubMed: 30653276
DOI: 10.1002/pro.3574 -
European Journal of Biochemistry Feb 1990Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was...
Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 micrograms/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2(+)-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide-gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.
Topics: Amino Acid Sequence; Amino Acids; Animals; DNA; Electrophoresis, Polyacrylamide Gel; Gene Expression; Genetic Vectors; Glycoproteins; Glycoside Hydrolases; Humans; Mice; Molecular Sequence Data; Plasmids; Protein Processing, Post-Translational; Protein S; Recombinant Proteins; Structure-Activity Relationship; Transfection
PubMed: 2137411
DOI: 10.1111/j.1432-1033.1990.tb15361.x -
FEBS Letters Nov 1995A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast. Here we describe the... (Comparative Study)
Comparative Study
A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast. Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S-transferase in E. coli and its kinetic characterisation. Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M(r) protein phosphatase previously known to exist only in mammalian species. Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M(r) protein tyrosine phosphatases present in mammalian tissues. These differing kinetic characteristics are mainly due to the sequence 45-56 that is spatially close to the active site pocket.
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Cloning, Molecular; Cytosol; Escherichia coli; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Proteins; Oligodeoxyribonucleotides; Protein Tyrosine Phosphatases; RNA-Binding Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Transcription Factors
PubMed: 7498507
DOI: 10.1016/0014-5793(95)01220-9 -
The EMBO Journal Aug 1993The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast...
The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.
Topics: Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Cell Division; Centrifugation; Chromatography, Affinity; Cloning, Molecular; DNA, Fungal; Electrophoresis, Polyacrylamide Gel; Epitopes; Fungal Proteins; Immunoglobulin G; Membrane Glycoproteins; Membrane Proteins; Molecular Sequence Data; Nuclear Pore Complex Proteins; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 7688296
DOI: 10.1002/j.1460-2075.1993.tb05975.x -
Nucleic Acids Research Feb 1983E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S. Wickner, I.Berkower, M.Wright, and J. Hurwitz (1973) Proc. Natl. Acad....
E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S. Wickner, I.Berkower, M.Wright, and J. Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S. Wickner and J. Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.
Topics: Bacterial Proteins; Escherichia coli; Macromolecular Substances; Molecular Weight
PubMed: 6298736
DOI: 10.1093/nar/11.4.987