共查询到20条相似文献,搜索用时 15 毫秒
1.
Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody 总被引:1,自引:0,他引:1
A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73. 相似文献
2.
Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the VH and VL gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (KD =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein. 相似文献
3.
Sean A. Gray Kris M. Weigel Keith D. Miller Joseph Ndung'u Philippe Büscher Thao Tran Cheryl Baird Gerard A. Cangelosi 《Biotechnology and bioengineering》2010,105(5):973-981
Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast‐display libraries. Yeast‐display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non‐immune libraries is the conversion of highly active yeast‐displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast‐display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast‐displayed and ‐secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast‐displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast‐displayed scFv that bind non‐overlapping or non‐competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast‐displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast‐displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast‐display libraries. Biotechnol. Bioeng. 2010;105: 973–981. © 2009 Wiley Periodicals, Inc. 相似文献
4.
Miller KD Weaver-Feldhaus J Gray SA Siegel RW Feldhaus MJ 《Protein expression and purification》2005,42(2):221-267
Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships. 相似文献
5.
The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay. 相似文献
6.
Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I 总被引:4,自引:0,他引:4
An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies. 相似文献
7.
Construction of a diabody (small recombinant bispecific antibody) using a refolding system 总被引:8,自引:0,他引:8
Takemura S Asano R Tsumoto K Ebara S Sakurai N Katayose Y Kodama H Yoshida H Suzuki M Imai K Matsuno S Kudo T Kumagai I 《Protein engineering》2000,13(8):583-588
Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system. 相似文献
8.
Seurynck-Servoss SL Baird CL Miller KD Pefaur NB Gonzalez RM Apiyo DO Engelmann HE Srivastava S Kagan J Rodland KD Zangar RC 《Proteomics》2008,8(11):2199-2210
Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems. 相似文献
9.
Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment 总被引:1,自引:0,他引:1
Asano R Kudo T Nishimura Y Makabe K Hayashi H Suzuki M Tsumoto K Kumagai I 《Journal of biochemistry》2002,132(6):903-909
Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody). 相似文献
10.
Peterson NC 《ILAR journal / National Research Council, Institute of Laboratory Animal Resources》2005,46(3):314-319
The ability to produce antibodies that are directed against specific antigens has played a crucial role in advancing scientific discoveries. Recombinant technologies have extended the application of antibodies beyond the research laboratory and into the clinic for the treatment of cancer and other diseases. Creative approaches using these technologies have been used to reduce the antibody to its minimal functional size, and/or make them bifunctional (immunotoxins), bispecific, or less immunoreactive (humanized). Additionally, mice that are engineered to generate antibodies of human genomic origin have been used to produce therapeutic antibodies and are being further developed. As the research and clinical demands for antibodies continue to increase, the development of improved resources (cell lines and animals) to improve production efficiency, generate larger repertoires, and deliver greater yields of antibodies is being explored, and advances in this area are discussed further in this review. 相似文献
11.
Gunsup Lee Jaelim Yu Seungchan Cho Sung-June Byun Dae Hyun Kim Taek-Kyun Lee Myung-Hee Kwon Sukchan Lee 《PLoS pathogens》2014,10(6)
Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system. 相似文献
12.
为探讨肝癌特异性鼠源及其人源化单链抗体基因的表达策略并比较二者的结合能力,在3种载体中分别以融合、分泌及胞内表达的方式进行了研究;对复性后的单链抗体以抗原捕获ELISA法进行检测。结果表明,在3种载体中表达的鼠源及人源化单链抗体都是包含体,诱导物浓度及培养温度不影响表达形式;抗原捕获细胞ELISA表明人源化的单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是:在大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设计所采取的人源化方案没有影响到鼠源抗体的CDRs的天然构象,表达的人源化单链抗体提供了免疫原性评价及临床应用的基础。 相似文献
13.
Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases. 相似文献
14.
The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research. 相似文献
15.
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
16.
The aim of the present study was to discover distinct human MAbs to RV with high neutralizing potency and a broad neutralization spectrum. A phage display technology was used to produce human scFv to G5, a conserved linear neutralization epitope on Gp of RV. A phage display scFv library with 6 x 10(7) members was constructed and the phage-scFv with 'antigen-binding' activities were selected with synthetic peptide G5-24. The obtained scFv genes were cloned into pET22b(+)/BL21(DE3) and from this we prepared purified scFv fragments. The assay of the specificity characteristics and neutralization capacity showed that two distinct clones with new human immunoglobulin V genes can recognize G5 specifically as well as neutralize different RV strains. They have potential for inclusion in an antibodies combination aimed for use in rabies PEP. 相似文献
17.
Arcangeli C Cantale C Galeffi P Gianese G Paparcone R Rosato V 《Journal of biomolecular structure & dynamics》2008,26(1):35-48
Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti-HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies. 相似文献
18.
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
19.
High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived
from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins
is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing
partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts
than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this
purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas
are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins
that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large,
human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55
distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed
for binding specificity and soluble scFv can be readily produced and purified via the appended His6 epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex
formation and are suitable for co-crystallization trials. 相似文献
20.
Sakamoto S Tanizaki Y Pongkitwitoon B Tanaka H Morimoto S 《Protein expression and purification》2011,77(1):124-130
A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. 相似文献