共查询到20条相似文献,搜索用时 22 毫秒
1.
Hong Wang Jinyi Yang Xixia Liu Yan Liang Hongtao Lei Yudong Shen Xiaoyan Xu Yuanming Sun Zhenlin Xu Yongsheng He 《Biotechnology progress》2009,25(4):1018-1024
Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly4Ser)3. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC50 value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
2.
GFP‐SCFV: Expression and possible applications as a tool for experimental investigations of atherosclerosis 下载免费PDF全文
Aldacilene Souza Silva Dulcineia Saes Parra Abdalla Vitor Renaux Hering Mario José Politi Andrea Queiroz Maranhão 《Biotechnology progress》2014,30(5):1206-1213
Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(?), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti‐LDL(?) scFv, previously obtained from a hybridoma secreting an anti‐LDL(?) monoclonal antibody, was inserted into the bacterial vector pET‐28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP‐scFv was assessed by ELISA, which determined its affinity for LDL(?) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(?). In conclusion, our data suggest that the anti‐LDL(?) GFP‐scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1206–1213, 2014 相似文献
3.
Ling Ding Mark Azam Yu-Huei Lin James Sheridan Shuanghong Wei Gigi Gupta Rakesh K Singh Michelle H Pauling Waihei Chu Antares Tran Nai-Xuan Yu Jiefeng Hu Wei Wang Hao Long Dong Xiang Li Zhu Shao-Bing Hua 《Protein science : a publication of the Protein Society》2010,19(10):1957-1966
We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single‐chain variable fragment (scFv) antibody library was constructed in a yeast two‐hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin‐8 (hIL8) into the yeast two‐hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error‐prone PCR of the scFv sequence followed by additional rounds of yeast two‐hybrid screening. The scFv antibodies of both primary and affinity‐matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost. 相似文献
4.
Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichia
coli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110 kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human. 相似文献
5.
Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of
recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment
(scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly
in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related
modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain.
The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The
fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease
to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance
liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of
the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight
mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit
VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study
allows convenient high yield and easy purification of recombinant scFv with native sequences. 相似文献
6.
Junjie Xu Jian Song Fei Yan Huiying Chu Jixun Luo Yongshan Zhao Xu Cheng Guimin Luo Qingchuan Zheng Jingyan Wei 《Journal of molecular recognition : JMR》2009,22(4):293-300
Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni2+‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
7.
Wu G Wang X Deng X Wu P Xue X Yan X Wang X 《Applied microbiology and biotechnology》2012,93(6):2475-2482
This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor
(anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in
Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification.
Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence
experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody
through inhibiting the phosphorylation of Akt. 相似文献
8.
Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein 下载免费PDF全文
Siew Wen Leong Theam Soon Lim Asma Ismail Yee Siew Choong 《Journal of molecular recognition : JMR》2018,31(5)
With the development of de novo binders for protein targets from non‐related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single‐chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking “disembodied” amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein‐antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen‐based detection agents for typhoid diagnostics. 相似文献
9.
Damilola D. Phillips David N. Garboczi Kavita Singh Zonglin Hu Stephen H. Leppla Clinton E. Leysath 《Journal of molecular recognition : JMR》2013,26(8):376-381
The monoclonal antibody S9.6 binds DNA–RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R‐loops. A single‐chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA–RNA and, surprisingly, 2.7 nM for RNA–RNA hybrids that are AU‐rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA–RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA. 相似文献
10.
Lin Wan Shengyun Zhu Jian Zhu Hao Yang Shengfu Li Youping Li Jingqiu Cheng Xiaofeng Lu 《Applied microbiology and biotechnology》2013,97(9):3855-3863
Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60–70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders. 相似文献
11.
Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells 下载免费PDF全文
Guillaume Freund Dominique Desplancq Audrey Stoessel Robin Weinsanto Annie‐Paule Sibler Gautier Robin Pierre Martineau Pascal Didier Jérôme Wagner Etienne Weiss 《Journal of molecular recognition : JMR》2014,27(9):549-558
Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
12.
Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display. 相似文献
13.
M. Ribbert A. Wolters S. Barth M. Stoecker A. Schaeffer R. Fischer R. Finnern 《Journal of Phytopathology》2007,155(3):170-177
We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V‐gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non‐germinated ascospores. The binding of selected phage antibodies was monitored by enzyme‐linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross‐reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage‐derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers. 相似文献
14.
Sean A. Gray John R. Barr Suzanne R. Kalb James D. Marks Cheryl L. Baird Gerard A. Cangelosi Keith D. Miller Michael J. Feldhaus 《Biotechnology and bioengineering》2011,108(10):2456-2467
A non‐immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody‐mediated labeling strategy was used in which antigen‐binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full‐length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep‐MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A‐specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep‐MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep‐MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456–2467. © 2011 Wiley Periodicals, Inc. 相似文献
15.
Jonathan Hurtado Jake Stahnke Anja Fuchs Erin Mehlhop Sergey Gorlatov Andreas Loos Michael S. Diamond Qiang Chen 《Plant biotechnology journal》2014,12(8):1098-1107
Previously, our group engineered a plant‐derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single‐chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv‐CH). pE16 and pE16scFv‐CH were expressed and assembled efficiently in Nicotiana benthamiana ?XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ?XF plant‐derived pE16scFv‐CH (?XFpE16scFv‐CH) and pE16 (?XFpE16) both displayed a mammalian glycosylation profile. ?XFpE16 and ?XFpE16scFv‐CH demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (mE16). A single dose of ?XFpE16 or ?XFpE16scFv‐CH protected mice against WNV‐induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases. 相似文献
16.
Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase 总被引:3,自引:0,他引:3
Petrov K Dion M Hoffmann L Dintinger T Defontaine A Tellier C 《Journal of molecular biology》2004,341(4):1039-1048
The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity. 相似文献
17.
Howell S Kenmore M Kirkland M Badley RA 《Journal of molecular recognition : JMR》1998,11(1-6):200-203
There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N-hydroxysuccinimide (NHS) and N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti-IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed. 相似文献
18.
Marty C Scheidegger P Ballmer-Hofer K Klemenz R Schwendener RA 《Protein expression and purification》2001,21(1):156-164
The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments. 相似文献
19.
Niemi MH Takkinen K Amundsen LK Söderlund H Rouvinen J Höyhtyä M 《Journal of molecular recognition : JMR》2011,24(2):209-219
A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 108 clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
20.
An anti-chlorpropham single-chain variable-fragment (scFv) gene was introduced into Arabidopsis in a manner to express the antibody fragment in each of four different subcellular compartments. The accumulation of scFv in transgenic plants was detected by targeting the fragment in the endoplasmic reticulum or apoplastic space, or by expressing the fragment as a glycosylphosphatidylinositol-anchored protein, while no accumulation could be detected by targeting the fragment in the cytosol. Transgenic plants accumulating the scFv gene at a high level in the endoplasmic reticulum had enhanced tolerance to chlorpropham in comparison with the non-transformants. 相似文献