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1.
We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics. 相似文献
2.
A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. 总被引:15,自引:1,他引:14
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K Büssow D Cahill W Nietfeld D Bancroft E Scherzinger H Lehrach G Walter 《Nucleic acids research》1998,26(21):5007-5008
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins. 相似文献
3.
Vinita Puri Emily Streaker Ponraj Prabakaran Zhongyu Zhu Dimiter S. Dimitrov 《MABS-AUSTIN》2013,5(4):533-539
Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a yeast display naïve antibody library. All the yeast display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins. 相似文献
4.
Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3 总被引:7,自引:0,他引:7
Rauchenberger R Borges E Thomassen-Wolf E Rom E Adar R Yaniv Y Malka M Chumakov I Kotzer S Resnitzky D Knappik A Reiffert S Prassler J Jury K Waldherr D Bauer S Kretzschmar T Yayon A Rothe C 《The Journal of biological chemistry》2003,278(40):38194-38205
The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., W?lle, J., Plückthun, A., and Virnek?s, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications. 相似文献
5.
Cajsa Älgenäs Charlotta Agaton Linn Fagerberg Anna Asplund Lisa Björling Erik Björling Caroline Kampf Emma Lundberg Peter Nilsson Anja Persson Kenneth Wester Fredrik Pontén Henrik Wernérus Mathias Uhlén Jenny Ottosson Takanen Sophia Hober 《Biotechnology journal》2014,9(3):435-445
An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well‐designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application‐specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full‐length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full‐length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context‐ and application‐dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross‐reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data. 相似文献
6.
Schuster M Wasserbauer E Einhauer A Ortner C Jungbauer A Hammerschmid F Werner G 《Journal of biomolecular screening》2000,5(2):89-97
Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats. 相似文献
7.
Ronald E. Ferrando Kim Newton Felix Chu Joshua D. Webster Dorothy M. French 《The journal of histochemistry and cytochemistry》2015,63(4):244-255
With recent advances in immunohistochemical (IHC) techniques, immunohistochemistry now plays a more important role in research, especially in mouse models where characterization of cellular patterns of protein expression has become critical. Even with these recent advances, a paucity of IHC quality antibodies for some proteins still exists. To address this, we have developed a novel IHC assay that utilizes a commercially available goat anti-DDDDK peptide polyclonal antibody on paraffin-embedded tissues from knock-in mice expressing proteins of interest tagged with a 3×FLAG epitope at physiologically relevant levels. Focusing on two 3×FLAG-tagged proteins for which specific antibodies were available, USP48 and RIPK3, we were able to validate our anti-DDDDK assay by comparing the IHC directed against the actual proteins to the anti-DDDDK IHC assay, which recognizes the FLAG epitope. We were also able to detect a third 3×FLAG-tagged protein, BAP1, for which quality reagents were not available. This universal IHC method will enable researchers to characterize the expression patterns of proteins of interest when specific antibodies are lacking. 相似文献
8.
Thota A Karajgikar M Duan W Gabril MY Chan FL Wong YC Sakai H Chin JL Moussa M Xuan JW 《Journal of cellular biochemistry》2003,88(5):999-1011
Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model. 相似文献
9.
Fahd Al-Mulla Mahera Abdulrahman Govindarajulu Varadharaj Nadeem Akhter Jehoram T Anim 《The journal of histochemistry and cytochemistry》2005,53(5):621-629
Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard. 相似文献
10.
Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen 总被引:3,自引:0,他引:3
In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection. 相似文献
11.
Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin 总被引:5,自引:4,他引:1
Sheldon L. Miller David Pleasure † Meenhard Herlyn Barbara Atkinson ‡ Carolyn Ernst ‡ Thomas G. Tachovsky Lynn Baird 《Journal of neurochemistry》1984,43(2):394-400
Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low. 相似文献
12.
Furuta M Ito T Eguchi C Tanaka T Wakabayashi-Takai E Kaneko K 《Journal of biochemistry》2002,132(2):245-251
We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble "lipid rafts" as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions. 相似文献
13.
Panavas T Lu J Liu X Winkis AM Powers G Naso MF Amegadzie B 《Protein expression and purification》2011,79(1):7-15
Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity. 相似文献
14.
Constantina D Petraki Vassiliki N Karavana Liu-Ying Luo Eleftherios P Diamandis 《The journal of histochemistry and cytochemistry》2002,50(9):1247-1261
The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern. 相似文献
15.
Mucin-1 has proven to be a suitable target for antibody-based diagnosis and therapy of certain tumours, but no appropriate human antibody or antibody fragment displaying slow dissociation rate kinetics against this target is available. Since a rapid dissociation character prevents an antibody fragment from remaining at the site of the antigen, this fact may prevent the successful application of a human mucin-1 specific antibody in diagnosis and therapy. We have now used iterative antibody libraries to evolve a human antibody fragment originally obtained from a na?ve antibody library. A strategy was devised whereby molecular variants displaying slow dissociation kinetics against the repetitive mucin-1 tumour-associated antigen can be selected in vitro. The evolved clones, that allowed for a reduced dissociation from the tumour antigen, carried substitutions in the outer parts of the binding site. This demonstrated the ability of this in vitro evolution technique to mimic the process whereby antibodies evolve in vivo. We have thus devised a strategy through which molecular variants displaying slow dissociation from a repetitive target like the mucin-1 tumour-associated antigen can be obtained in vitro. These or related molecules obtained by this approach will serve as a starting point for the development of fully human antibodies for use in mucin-1 specific tumour therapy of diagnosis. 相似文献
16.
Killeen GF Foy BD Frohn RH Impoinvil D Williams A Beier JC 《Bulletin of entomological research》2003,93(1):31-37
A high diversity library of recombinant human antibodies was selected on complex antigen mixtures from midguts of female Anopheles gambiae Giles. The library of phage-displayed single chain variable region fragment constructs, derived from beta-lymphocyte mRNA of na?ve human donors, was repeatedly selected and reamplified on the insoluble fraction of midgut homogenates. Five rounds of panning yielded only one midgut-specific clone, which predominated the resulting antibody panel. In A. gambiae, the epitope was found throughout the tissues of females but was absent from the midgut of males. The cognate antigen proved to be detergent soluble but very sensitive to denaturation and could not be isolated or identified by Western blot of native electrophoresis gels or by immunoprecipitation. Nevertheless, immunohistology revealed that this sex-specific epitope is associated with the lumenal side of the midgut. Severe bottlenecking may limit the utility of phage display selection from na?ve libraries for generating diverse panels of antibodies against complex mixtures of antigens from insect tissues. These results suggest that the selection of sufficiently diverse antibody panels, from which mosquitocidal or malaria transmission-blocking antibodies can be isolated, may require improved selection methods or specifically enriched pre-immunized libraries. 相似文献
17.
Emil Bujak Francesca Pretto Danilo Ritz Laura Gualandi Sarah Wulhfard Dario Neri 《Experimental cell research》2014
There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. 相似文献
18.
Human endogenous retrovirus K10: expression of Gag protein and detection of antibodies in patients with seminomas. 总被引:14,自引:8,他引:6
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M Sauter S Schommer E Kremmer K Remberger G Dlken I Lemm M Buck B Best D Neumann-Haefelin N Mueller-Lantzsch 《Journal of virology》1995,69(1):414-421
The human endogenous retrovirus K10 (HERV-K10) has been identified in the human genome by its homology to retroviruses of other vertebrates (M. Ono, T. Yasunaga, T. Miyata, and H. Ushikubo, J. Virol. 60:589-598, 1986). Using PCR amplification, DNA cloning, sequencing, and procaryotic expression, we were able to demonstrate that HERV-K10 encodes a 73-kDa protein which was processed by a HERV-K10-encoded protease to yield proteins p22/p26, p30, and p15/16. Analysis of the teratocarcinoma cell line Tera 1 or tumor tissues by immunoblotting demonstrated that the 80-kDa polyprotein of HERV-K10 gag and a processed protein of 39 kDa were expressed. In addition, a major protein of 39 kDa and additional species of 30, 22, 19, and 17 kDa could be detected in the supernatant of Tera 1 cells, suggesting that HERV-K10 Gag proteins are either secreted or processed to probably incomplete viral particles. In addition, the gag gene of HERV-K10 was expressed in the baculovirus system. Using this recombinant system to test antisera from patients with different diseases and healthy individuals, we were able to detect antibodies against the N-terminal part of HERV-K10 Gag in 2 to 4% of groups of tumor patients with titers ranging between 1:80 and 1:640, while approximately 0.1 to 0.5% of healthy individuals exhibited antibodies with lower titers. In contrast, patients with seminoma had antibody titers in the range of 1:2,560 at the time when the tumor was detected. Immunohistochemistry using specific rabbit sera or monoclonal antibodies against HERV-K10 Gag revealed that the Gag protein is expressed in the cytoplasm of the tumor cells. Furthermore, an 80-kDa protein corresponding to the HERV-K10 Gag polyprotein could be detected in tumor biopsies. For the first time, these data indicate that HERV-K10 Gag proteins are synthesized in seminoma cells and tumors exhibit relatively high antibody titers against Gag. So far, no information on which role HERV-K10 plays in the development of this tumor exists. 相似文献
19.
ABSTRACT: BACKGROUND: Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. RESULTS: In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. CONCLUSIONS: Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins. 相似文献
20.
Santos Michael O. Crosby William L. Winkel Brenda S.J. 《Molecular breeding : new strategies in plant improvement》2004,13(4):333-343
The utility of recombinant antibodies for immunomodulation of plant metabolism was tested using the Arabidopsis flavonoid biosynthetic pathway as a target. Two genes encoding antibodies against chalcone isomerase (CHI) were isolated from a human synthetic single chain variable fragment (scFv) library and expressed in the cytoplasm of transgenic plants. In one line, low-level expression of the scFv resulted in reduced visible pigmentation in seedlings as well as lower accumulation of phenolics in plants throughout development. Surprisingly, other transgenic lines with much higher expression of the same antibody showed no phenotype. Protein mobility shift assays indicated that the scFv is bound to the target enzyme, but only in the affected plants. This work shows that recombinant scFv antibody technology offers a viable approach to disrupting protein activity in plants, but that further refinement is required before it will be of general utility for metabolic engineering and other antibody-based applications in plants. 相似文献