McrBs, a modulator peptide for McrBC activity.

McrBC is a methylation-dependent endonuclease from Escherichia coli K-12. The enzyme recognizes DNA with modified cytosines preceded by a purine. McrBC restricts DNA that contains at least two methylated recognition sites separated by 40-80 bp. Two gene products, McrBL and McrBs, are produced from the mcrB gene and one, McrC, from the mcrC gene. DNA cleavage in vitro requires McrBL, McrC, GTP and Mg2+. We found that DNA cleavage was optimal at a ratio of 3-5 McrBL per molecule of McrC, suggesting that formation of a multisubunit complex with several molecules of McrBL is required for cleavage. To understand the role of McrBs, we have purified the protein and analyzed its role in vitro. At the optimal ratio of 3-5 McrBL per molecule of McrC, McrBs acted as an inhibitor of DNA cleavage. Inhibition was due to sequestration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent. If McrC was in excess, a condition resulting in suboptimal DNA cleavage, addition of McrBs enhanced DNA cleavage, presumably due to sequestration of excess McrC. We suggest that the role of McrBs is to modulate McrBC activity by binding to McrC.     

Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' R(m)C 3'. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB(L) binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg(2+). The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB(L)-binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPgammaS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.     

A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli

McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites". It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage. A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences. A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E. coli McrC. Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC. Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.     

McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.

Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis.     

The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat

The GTP-dependent restriction enzyme McrBC consists of two polypeptides: one (McrB) that is responsible for GTP binding and hydrolysis as well as DNA binding and another (McrC) that is responsible for DNA cleavage. It recognizes two methylated or hemimethylated RC sites (R(m)C) at a distance of approximately 30 to more than 2000 base pairs and cleaves the DNA close to one of the two R(m)C sites. This process is strictly coupled to GTP hydrolysis and involves the formation of high-molecular mass complexes. We show here using footprinting techniques, surface plasmon resonance, and scanning force microscopy experiments that in the absence of McrC, McrB binds to a single R(m)C site. If a second R(m)C site is present on the DNA, it is occupied independently by McrB. Whereas the DNA-binding domain of McrB forms 1:1 complexes with each R(m)C site and shows a clear footprint on both R(m)C sites, full-length McrB forms complexes with a stoichiometry of at least 4:1 at each R(m)C site, resulting in a slightly more extended footprint. In the presence of McrC, McrB forms high-molecular mass complexes of unknown stoichiometry, which are considerably larger than the complexes formed with McrB alone. In these complexes and when GTP is present, the DNA is cleaved next to one of the R(m)C sites at distances differing by one to five helical turns, suggesting that in the McrBC-DNA complex only a few topologically well-defined phosphodiester bonds of the DNA are accessible for the nucleolytic center of McrC.     

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.     

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

Defining the location and function of domains of McrB by deletion mutagenesis

The GTP-dependent restriction endonuclease McrBC of E. coli K12, which recognizes cytosine-methylated DNA, consists of two protein subunits, McrB and McrC. We have investigated the structural assignment and interdependence of the McrB subunit functions, namely (i) specific DNA recognition and (ii) GTP binding and hydrolysis. Extending earlier work, we have produced McrB variants comprising N- and C-terminal fragments. The variants McrB1-162 and McrB1-170 are still capable of specific DNA binding. McrB169-465 shows GTP binding and hydrolysis characteristics indistinguishable from full-length McrB as well as wild-type like interaction with McrC. Thus, DNA and GTP binding are spatially separated on the McrB molecule, and the respective domains function quite independently.     

Purification and Characterization of Biliverdin-binding Protein from Larval Hemolymph of the Swallowtail Butterfly,Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.     

Purification and DNA-binding properties of human papillomavirus type 16 E6 protein expressed in Escherichia coli

Unfused human papillomavirus type 16 (HPV 16) E6 protein was expressed in Escherichia coli using a lambda PL promoter system. The protein was isolated from the cells as inclusion bodies, extracted by 6 M guanidine-HCl, and purified by chromatography. The purified protein had high affinity to DNA and was demonstrated for the first time to bind to a specific sequence within the long control region of HPV 16.     

Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.

We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2- to 3-fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Function of MglA, a 22-kilodalton protein essential for gliding in Myxococcus xanthus.

Single mutations in the mglA gene in Myxococcus xanthus render cells incapable of gliding. The mglA strains are unique in that all other nonmotile strains of M. xanthus isolated are the result of at least two independent mutations in separate motility system genes. Translational fusions of trpE, or of lacZ, to mglA were constructed, and the resulting fusion polypeptides were used to generate antibodies. Antibodies specific to MglA protein were purified. Antibody-tagged MglA was found localized to the cytoplasm of M. xanthus cells both by fractionation of cell extracts and by electron microscopy of thin sections of whole cells. Four of the five mglA missense mutants tested failed to produce detectable levels of the MglA antigen in whole cell extracts. Nonmotile double mutants (A-S-), which have one mutation in a gene of system A and one mutation in a gene of system S, have the same phenotype as null mglA mutants but produce wild-type levels of MglA protein. MglA protein is conserved in all strains of myxobacteria tested. The amino acid sequence of MglA protein includes three sequence motifs characteristic of GDP/GTP-binding proteins. On the basis of its genetic properties, intracellular location, and amino acid sequence, it is argued that MglA protein is a regulator in the sequence of functions leading to cell movement.     

Effective method for purification of lysozyme from human urine

Lysozyme in the urine of a hemodialysis patient was purified in two steps: DEAE Sephadex chromatography followed by Sephacryl chromatography. The Sephacryl S-100 column chromatographed fraction showing lytic activity was proven to give one band on SDS-PAGE and to have a molecular mass of 14 500, in agreement with that of lysozyme. The N-terminal amino acid sequence of this purified protein was identical to that of lysozyme. These results indicate that the protein purified was indeed lysozyme. The specific affinity of lysozyme for Sephacryl S-100 may explain the greater purity of the same protein isolated by this method.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.