Streptococcus agalactiae GAPDH is a virulence-associated immunomodulatory protein

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.     

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.     

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.     

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody

Abstract A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells(SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei . The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotypy h), but not with cells or purified PS antigen from any other serotypes of the mutants group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that β-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the β-methylgalactopyranoside molecule.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.     

Purification of urokinase by monoclonal antibody affinity chromatography

Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity.     

Purification of human melanocytes by monoclonal antibody combined with Percoll gradients

Cultures of human melanocytes obtained by differential plating of human epidermal cells in 12-myristate 13-acetate (PMA) were purified using a monoclonal antibody R₂₄ which detects a restricted glycolipid antigen present on melanoma cells and melanocytes. Melanocytes were rosetted using protein A-conjugated human red blood cells and separated from non-rosetted ftbroblasts on discontinuous Percoll^™ gradients. 100% pure cultures of melanocytes obtained in this fashion were then successfully grown in tissue culture in the presence of PMA and cholera toxin for at least thirty passages (corresponding to approx. sixty cell doublings). We conclude that:

1. 1. Pure cultures of melanocytes are a prerequisite for the establishment of long-term cultures.

2. 2. Since most human melanomas express substantial levels of R₂₁ antigen, this method can be applied to purification of melanomas and can be easily adapted to separation of subpopulations of melanocytes and melanoma cells recognized by specific monoclonal antibodies.

     

Purification of bovine glia maturation factor and characterization with monoclonal antibody

Glia maturation factor (GMF) is purified 100 000-fold to apparent homogeneity from bovine brains by a procedure consisting of ammonium sulfate precipitation, column chromatography with diethylaminoethyl-Sephacel, Sephadex G-75, and hydroxylapatite, and a final step using C4 reverse-phase high-performance liquid chromatography. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 14 000 and an isoelectric point of pH 5.2. Purified GMF stimulates cultured astroblasts to proliferate and to grow out cell processes with half-maximal activity at 8 ng/mL. A monoclonal antibody raised against partially purified GMF adsorbs the activity of pure GMF and immunologically binds the putative GMF protein band.     

Purification of aromatic L-amino acid decarboxylase from bovine brain with a monoclonal antibody.

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5'-phosphate as a cofactor, both enzymes were similar.     

Pathogenesis of neonatal Streptococcus agalactiae infections

Streptococcus agalactiae is an important human pathogen causing severe neonatal infections. During the course of infection, S. agalactiae colonizes and invades a number of different host compartments. Bacterial molecules including the polysaccharide capsule, the hemolysin, the C5a peptidase, the C-proteins, the hyaluronate lyase and a number of unknown bacterial components determine the interaction with host tissues. This review summarizes our current knowledge about these interactions.     

Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.

A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.     

Purification of RNA using an anti-RNA monoclonal antibody.

Studies of the synthesis and modification of RNA employ many types of in vitro reactions. Often, the RNA product must be concentrated or purified away from other reaction components such as salts, unincorporated nucleotides, protein, or DNA. Here I describe an immunological approach suitable for the isolation of RNA from in vitro reactions. A variety of RNAs of differing size and nucleotide sequence were immunoprecipitated with a monoclonal antibody specific for RNA. RNA binding took place in seconds with nearly quantitative recoveries. Immunoprecipitation was more efficient than ethanol precipitation in removing unincorporated nucleotides. Proteins which do not bind to RNA remained soluble. The immunoprecipitated RNA sample was solubilized directly with a buffered solution suitable for gel electrophoresis under denaturing conditions. Thus, RNAs can be rapidly concentrated for electrophoresis in a single step. Antibody-RNA binding was reversible under nondenaturing conditions in the presence of excess rRNA. This procedure serves as a novel means of purifying RNA and RNA-binding proteins from in vitro reactions.     

日本血吸虫重组信号蛋白14—3—3的纯化及单克隆抗体的制备

纯化日本血吸虫（中国大陆株）重组信号蛋白（rSj14—3—3）,并制备其单克隆抗体。以纯化后的rsj14-3—3蛋白为抗原免疫Balb／c小鼠,用杂交瘤技术制备抗rSj14-3-3的单克隆抗体,并通过ELISA方法和Westernblotting测定抗体的效价与特异性。获得了大量高纯度的rSj14-3-3蛋白：筛选出了能够稳定分泌抗rSj14．3．3单抗的杂交瘤细胞株3H6。单抗亚型为IgG1。实验依靠大肠杆菌表达系统高效表达了rSj14—3—3蛋白,并利用该蛋白制备了单克隆抗体．可用于今后血吸虫病免疫诊断的实验研究。     

Characterization of Streptococcus agalactiae CAMP factor as a pore-forming toxin

A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12-15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action.     

Draft Genome Sequence of a Nonhemolytic Fish-Pathogenic Streptococcus agalactiae Strain

Streptococcus agalactiae is a significant Gram-positive bacterial pathogen of terrestrial and aquatic animals. A subpopulation of nonhemolytic strains which appear to be pathogenic only for poikilotherms exists. We report here the first draft genome sequence of a nonhemolytic S. agalactiae isolate recovered from a diseased fish.     

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.     

Production of a monoclonal antibody reacting with vimentin, a structural protein of intermediate filaments

This paper reports on the production of a monoclonal antibody (i-18) reacting with vimentin, the major structural component of intermediate filaments in cells of mesenchymal origin. The antibody was obtained following immunization with hamster fibroblasts and was selected for its ability to bind to the cytoskeleton fraction of the aforementioned cells. It decorated a perinuclear filamentous network characteristic of vimentin filaments in cells of mesenchymal origin of avian through human species. The specificity of the reagent was further ascertained on the basis of the sensitivity of the decorated filaments to colcemide. The strict antibody specificity for cells of mesenchymal versus epithelial origin was confirmed also in vivo on histological specimens from solid tissue. The i-18 monoclonal antibody precipitated a molecule of about 57 Kd from metabolically labelled cellular extracts. The broad cross-reactivity of this monoclonal antibody among different animal species, as well as its strict in vivo mesenchymal tissue specificity makes this antibody a useful reagent for both experimental and diagnostic purposes.