PURPOSE: Recombinant ScFv lym-1 was produced, using pET vector system for large scale production. METHODS: ScFv lym-1 gene inserted pET-22b (+) vector, was expressed in E.coli BL-21 strain. ScFv lym-1 antibody extracted from periplasm, was purified with His-Taq column. To evaluated immunoreactivity with Raji cell, ScFv lym-1 was labeled with I-125 and I-125 ScFv lym-1 was purified with desalting column. Raji cell was injected into the C57BR/cdJ SCID mice. Gamma camera imaging were taken time point at 1, 8, 24, and 48 hr with 8 mm pinhole collimator. RESULTS: An active scFv lym-1 could be produced in E.coli with soluble from using pET vector system. Immunoreactivity and affinity constant of IgG lym-1 were 54% and 1.83 x 10(9) M(-1), respectively, and those of scFv lym-1 were 53.7% and 1.46 x 10(9) M(-1), respectively. Biodistribution of I-125 scFv lym-1 antibody showed faster clearance in blood, spleen, kidney and than I-125 IgG lym-1 antibody. Gamma camera image of I-125 scFv lym-1 antibody showed faster clearance and tumor targeting liver than I-125 IgG lym-1 antibody. CONCLUSIONS: In vitro properties of scFv lym-1 were similar to those of IgG lym-1. ScFv lym-1 showed faster blood clearance than IgG lym-1. These results suggest that scFv lym-1 antibody can be useful for tumor imaging agent.
Animals ; Gamma Cameras ; Immunoglobulin G ; Kidney ; Liver ; Lymphoma* ; Mice ; Mice, SCID ; Periplasm ; Radionuclide Imaging ; Spleen

In our previous report an anti-HBsAg human monoclonal antibody was generated using antibody phage display library technique. Using pComb3 filamentous phagemid vector, Fab molecule was expressed in fusion form to a phage coat protein in the periplasm of E. coli. A clone of HBsAg binder was selected after panning and designated as B7. In order to select the clones with higher affinity and to examine which chain contributes most to the affinity of B7, the light and heavy chain of B7 was sequentially deleted and replaced with new library. HBsAg-binders were selected and tested by EIA (enzyme immunoassay). It was revealed that the affinity of B7 depends only on the heavy chain of Fd. B7 Fd was constructed without light chain and specificity and affinity was further confirmed by western blot analysis. This human monoclonal Fd antibody was found to react with d antigenic determinant of HBsAg as the original clone did. The nucleotide sequence analysis revealed that VH of B7 could be classified into Kabat's subgroup II and human IgG heavy chain family IV. The CH1 of B7 was IgG1.
Bacteriophages* ; Base Sequence ; Blotting, Western ; Clone Cells ; Hepatitis B Surface Antigens ; Humans ; Immunoglobulin G ; Periplasm ; Sensitivity and Specificity

Animals ; Cell Line ; Chloride Channels ; metabolism ; Epithelial Cells ; metabolism ; Escherichia coli ; metabolism ; Kidney ; cytology ; Osmotic Pressure ; Periplasm ; metabolism ; Swine

Helicobacter pylori are a capnophilic bacterium, which colonize gastric mucosa and are resistant to acidic and oxidative damage. Thiol-active proteins subserve redox functions in tolerating oxidative stress and environmental toxicants, such as hydrogen peroxide and hypochlorous acid. We analyzed disulfide-containing proteins of H. pylori strain 26695. Active disulfide-containing proteins were separated by thiol-affinity chromatography, displayed with two-dimensional electrophoresis (2-DE), and identified by MALDI-TOF-MS. Thirty-five putative disulfide proteins, including AhpC (HP1563), GroEL (HP0011), and FrdB (HP0191), were identified in this study. In addition, 4 disulfide proteins of HypB, FusA, TufB, and AhpC showed enhanced intensities in the periplasmic space when compared with the pellet, suggesting that these proteins might play roles in the first redox system against environmental oxidative stresses. Disulfide-containing proteins identified in this study will provide the standard landscape for constructing the proteome components responsible for redox regulation of H. pylori.
Chromatography ; Colon ; Electrophoresis ; Gastric Mucosa ; Helicobacter ; Helicobacter pylori ; Hydrogen Peroxide ; Hypochlorous Acid ; Oxidation-Reduction ; Oxidative Stress ; Periplasm ; Proteins ; Proteome ; Sprains and Strains

BACKGROUND: Chlamydia pneumoniae causes pneumonia and upper respiratory tract infection and has been recently reported to be associated with coronary atherosclerosis. The difference between C. pneumoniae and other Chlamydia spp. has been demonstrated by serologic study, DNA analysis and ultrastructural observation. However, studies concerning the developmental cycle of C. pneumoniae are relatively short. This study was conducted to investigate the morphological changes and developmental characteristics of C pneumoniae in the HeLa cell. METHODS: To observe the intracellular inclusion of C. pneumoniae, the cultured HeLa cell monolayer was stained with Jones' iodine and Giemsa. The ultrastructures were examined with an electron microscope at 6, 24, 48, 72 and 96 hr after inoculation of elementary bodies. RESULTS: The C. pneumoniae organisms which formed intracytoplasmic inclusion bodies in HeLa cells were negative on iodine stain. In Giemsa-stained preparation, the inclusion bodies of variable sizes with a bluish purple color were identified in the cytoplasm of infected HeLa cells. After 6 hrs of infection, the elementary bodies with electron-dense spicule shaped substance of C. pneumoniae were enclosed by the HeLa cell membrane and were taken the host cell by endocytosis. After 24 hrs of infection, the electron-dense material in the elementary bodies were disappearing and the elementary bodies were transforming into reticulate bodies. After 48 hrs of infection, the reticulate bodies of C. pneumoniae were seen dividing by binary fission. Small electron-dense round bodies(miniature bodies) appeared near completion of division. After 72 hrs of infection. about half of the reticulate bodies were transformed into elementary bodies. Newly formed elementary bodies had a pear-shaped structure and large periplasmic space. After 96 hrs of infection. mature elementary bodies with condensed electron-dense material and a rigid outer membrane were observed. Miniature bodies were located in the cytoplasm of the elementary bodies. CONCLUSIONS: These unique morphological changes in HeLa cell culture show the developmental characteristics of C. pneumoniae.
Chlamydia* ; Chlamydophila pneumoniae* ; Coronary Artery Disease ; Cytoplasm ; DNA ; Endocytosis ; HeLa Cells ; Humans ; Inclusion Bodies ; Iodine ; Membranes ; Periplasm ; Pneumonia ; Respiratory Tract Infections

Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with lambda Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in H2O2 containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to H2O2. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.
Bacteria ; Gene Silencing ; Molecular Chaperones ; NADPH Oxidase ; Nitric Oxide ; Periplasm ; Phagocytes ; Reactive Nitrogen Species ; Reactive Oxygen Species ; Recombinases ; RNA, Messenger ; Salmonella typhimurium ; Salmonella* ; Sigma Factor ; Spheroplasts

In this study, we are to produce the single chain variable fragment (scFv) antibodies against surface protein of hepatitis B virus (HBV) using antibody phage display technique. Balb/c mice were immunized with preS1 and cDNAs of heavy and light chains of splenic B cells from immunized mice were prepared using RT-PCR. Two cDNAs were linked with (64S) linker DNA under recombination PCR to produce single chain Fv DNA. After digestion of scFv DNA with Sp 1 and Not 1, the digested DNA was ligated into pCANTAB 5E and electroporated into E. coli XL1-Blue to prepare scFv-library. The size of library was 1 * 10' pfu/ml. Phage antibodies (phabs) against preS1 were rescued with M13K07 helper phages, and preS1-binders were selected through 3 times of panning using 96 well microtitre plates. Phage antibody clones were assayed directly for the ability to bind preS1 by ELISA. And then 7 phage antibody clones had high ELISA signals against preS1. Phabs from preS1-specific pMsc-17 had the strongest ELISA signal to preS1. Phabs from pMsc-17 were used for Western blot to preS1 and the results revealed that it was specific to preS1. To prepare the soluble scFv antibody, phabs from pMsc-17 were transfected into non-suppressor E. coli HB2151, and grown under 1 mM IPTG. Soluble scFv antibody was mainly accumulated in the periplasmic space, but small amount of antibody was secreted into culture media.
Animals ; Antibodies ; B-Lymphocytes ; Bacteriophages* ; Blotting, Western ; Cell Surface Display Techniques ; Clone Cells ; Culture Media ; Digestion ; DNA ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Isopropyl Thiogalactoside ; Mice* ; Periplasm ; Polymerase Chain Reaction ; Recombination, Genetic ; Single-Chain Antibodies*