TY - JOUR A1 - VanderVen, Peter F. M. A1 - Ehler, Elisabeth A1 - Vakeel, Padmanabhan A1 - Eulitz, Stefan A1 - Schenk, Jörg A. A1 - Milting, Hendrik A1 - Micheel, Burkhard A1 - Fürst, Dieter Oswald T1 - Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/ VASP N2 - Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z- discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly. (c) 2006 Elsevier Inc. All rights reserved Y1 - 2006 U6 - https://doi.org/10.1016/j.yexcr.2006.03.015 ER - TY - JOUR A1 - Stuckas, Heiko A1 - Messerschmidt, Katrin A1 - Putzler, Sascha A1 - Baumann, Otto A1 - Schenk, Jörg A. A1 - Tiedemann, Ralph A1 - Micheel, Burkhard T1 - Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production N2 - The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/37692 U6 - https://doi.org/10.1002/Mrd.20916 SN - 1040-452X ER - TY - JOUR A1 - Stech, Marlitt A1 - Merk, Helmut A1 - Schenk, Jörg A. A1 - Stöcklein, Walter F. M. A1 - Wüstenhagen, Doreen Anja A1 - Micheel, Burkhard A1 - Duschl, Claus A1 - Bier, Frank Fabian A1 - Kubick, Stefan T1 - Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system JF - Journal of biotechnology N2 - Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner. KW - Cell-free KW - In vitro translation KW - Single chain antibody (scFv) KW - Insect lysate KW - Surface plasmon resonance Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2012.08.020 SN - 0168-1656 VL - 164 IS - 2 SP - 220 EP - 231 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Sheriff, Ahmed A1 - Vogt, B. A1 - Baumgart, Martin A1 - Montag, C. A1 - Hollenbach, B. A1 - Schenk, Jörg A. A1 - Ulrich, J. A1 - Ellias, F. A1 - Micheel, Burkhard T1 - Intracellular capture of B7 in antigen-presenting cells reduces costimulatory activity N2 - CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells. Y1 - 2003 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-47T23XK- 8&_coverDate=02%2F21%2F2003&_alid=268969757&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6713&_sort=d&view=c&_acct=C000053886&_v e ER - TY - JOUR A1 - Sellrie, Frank A1 - Schenk, Jörg A. A1 - Behrsing, Olaf A1 - Drechsel, Oliver A1 - Micheel, Burkhard T1 - Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma N2 - Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8. Y1 - 2007 UR - http://www.jbmb.or.kr/fulltext/jbmb/view.php?vol=40&page=875 ER - TY - JOUR A1 - Sellrie, Frank A1 - Schenk, Jörg A. A1 - Behrsing, Olaf A1 - Bottger, Volker A1 - Micheel, Burkhard T1 - A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer N2 - Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope. Y1 - 2002 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-44MGNDF- 1&_coverDate=03%2F01%2F2002&_alid=268992656&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4931&_sort=d&view=c&_acct=C000053886&_v e ER - TY - THES A1 - Schreiber, J. A1 - Stahn, R. A1 - Schenk, Jörg A. A1 - Karsten, U. A1 - Pecher, Gabriele T1 - Binding of tumor antigen mucin (MUC1) derived peptides to the heat shock protein DnaK Y1 - 2000 ER - TY - JOUR A1 - Schlag, Peter M. A1 - Osterziel, Karl Joseph A1 - Özcelik, Cemil A1 - Scherneck, Siegfried A1 - Wenzel, Katrin A1 - Daskalow, Katjana A1 - Herse, Florian A1 - Seitz, Susanne A1 - Zacharias, Ute A1 - Schenk, Jörg A. A1 - Schulz, Herbert A1 - Hübner, Norbert A1 - Micheel, Burkhard T1 - The protein phosphatase 1 inhibitor KEPI is down regulated in breast cancer cell lines and tissues and involved in the regulation of the tumour suppressor EGR1 via the MEK-ERK pathway N2 - KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway. Y1 - 2008 UR - http://www.atypon-link.com/doi/abs/10.1515/BC.2007.062 ER - TY - JOUR A1 - Schenk, Jörg A. A1 - Sellrie, Frank A1 - Böttger, Volker A1 - Micheel, Burkhard A1 - Stöcklein, Walter F. M. T1 - Generation and application of a fluorescein-specific single chain antibody N2 - A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface. Y1 - 2007 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRJ-4P3DY33- 1&_user=1584062&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053886&_version=1&_urlVersion=0&_userid=1584062&md5=e 4 ER - TY - JOUR A1 - Schenk, Jörg A. A1 - Matyssek, Franziska A1 - Micheel, Burkhard T1 - Interleukin 4 increases the antibody response against Rubisco in mice N2 - The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse. Y1 - 2004 ER - TY - JOUR A1 - Schenk, Jörg A. A1 - Fettke, Jörg A1 - Lenz, Christine A1 - Albers, Katharina A1 - Mallwitz, Frank A1 - Gajovic-Eichelmann, Nenad A1 - Ehrentreich-Förster, Eva A1 - Kusch, Emely A1 - Sellrie, Frank T1 - Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G JF - Journal of biotechnology N2 - The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem. KW - Hybridoma KW - SLPI KW - Protein G KW - Progesterone KW - Serum-free Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2011.12.025 SN - 0168-1656 VL - 158 IS - 1-2 SP - 34 EP - 35 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Schenk, Jörg A. T1 - Two Hybrid cDNA Cloning Y1 - 2002 ER - TY - JOUR A1 - Rohde, M. A1 - Schenk, Jörg A. A1 - Heymann, Stephan A1 - Behrsing, Olaf A1 - Scharte, Gudrun A1 - Kempter, Gerhard A1 - Woller, Jochen A1 - Höhne, Wolfgang A1 - Warsinke, Axel A1 - Micheel, Burkhard T1 - Production and characterization of monoclonal antibodeis against urea derivatives Y1 - 1998 ER - TY - JOUR A1 - Pecher, Gabriele A1 - Spahn, Gunter A1 - Schirrmann, Thomas A1 - Kulbe, Hagen A1 - Ziegner, Maja A1 - Schenk, Jörg A. A1 - Sandig, Volker T1 - Mucin gene (MUC1) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus N2 - BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC. Y1 - 2001 SN - 0250-7005 ER - TY - JOUR A1 - Pecher, Gabriele A1 - Schirrmann, Thomas A1 - Kaiser, Lothar A1 - Schenk, Jörg A. T1 - Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application N2 - Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials. Y1 - 2001 UR - http://www.babonline.org/bab/034/0161/bab0340161.htm ER - TY - JOUR A1 - Pecher, Gabriele A1 - Harnack, U. A1 - Gunther, M. A1 - Hummel, M. A1 - Fichtner, I. A1 - Schenk, Jörg A. T1 - Generation of an immortalized human CD4+ T cell clone inhibiting tumor growth in mice. N2 - Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the alpha/beta-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy. Y1 - 2001 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-45S4JD7- VM&_coverDate=05%2F18%2F2001&_alid=268965202&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6713&_sort=d&view=c&_acct=C000053886&_ v ER - TY - JOUR A1 - Lübbe, L. A1 - Schenk, Jörg A. A1 - Naundorf, H. A1 - Karsten, U. A1 - Wunderlich, V. T1 - Reverse transformation of human mammary carcinoma cells Y1 - 1999 ER - TY - JOUR A1 - Lawatscheck, Robert A1 - Aleksaite, Egle A1 - Schenk, Jörg A. A1 - Micheel, Burkhard A1 - Jandrig, Burkhard A1 - Holland, Gudrun A1 - Sasnauskas, Kestutius A1 - Gedvilaite, Alma A1 - Ulrich, Rainer Günter T1 - Chimeric polyomavirus-derived virus-like particles : the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence N2 - We inserted the sequence of the carcinoembryonic antigen-derived T cell epitope CAP-1-6D (CEA) into different positions of the hamster polyomavirus major capsid protein VP1. Independently from additional flanking linkers, yeast- expressed VP1 proteins harboring the CEA insertion between VP1 amino acid residues 80 and 89 (site 1) or 288 and 295 (site 4) or simultaneously at both positions assembled to chimeric virus-like particles (VLPs). BALB/c mice immunized with adjuvant-free VLPs developed VP1- and epitope-specific antibodies. The level of the CEA-specific antibody response was determined by the insertion site, the number of inserts, and the flanking linker. The strongest CEA-specific antibody response was observed in mice immunized with VP1 proteins harboring the CEA insert at site 1. Moreover, the CEA- specific antibodies in these mice were still detectable 6 mo after the final booster immunization. Our results indicate that hamster polyomavirus-derived VLPs represent a highly immunogenic carrier for foreign insertions that might be useful for clinical and therapeutic applications. Y1 - 2007 UR - http://www.liebertonline.com/vim U6 - https://doi.org/10.1089/vim.2007.0023 SN - 0882-8245 ER - TY - JOUR A1 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, Kristin A1 - Schenk, Jörg A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf J. T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA JF - Journal of immunological methods N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA Y1 - 2014 U6 - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Groth, Detlef A1 - Reszka, R. A1 - Schenk, Jörg A. T1 - Polyethylene glycol-mediated transformation of escherichia coli is increased by room temperature incubation Y1 - 1996 ER -