scholarly journals Strategies to improve scFvs as crystallization chaperones suggested by analysis of a complex with the human PHD-bromodomain SP140

2019 ◽  
Author(s):  
Michael Fairhead ◽  
Charlotta Preger ◽  
Edvard Wigren ◽  
Claire Strain-Damerell ◽  
Elena Ossipova ◽  
...  
Keyword(s):  
Structural Biology ◽  
Crystal Packing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Complex Structures ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Disease States ◽  
Display Technology

AbstractAntibody fragments have great potential as crystallization chaperones for structural biology due to their ability to either stabilise targets, trap certain conformations and/or promote crystal packing. Here we present an example of using a single-chain variable fragment (scFv) to determine the previously unsolved structure of the multidomain protein SP140. This nuclear leukocyte-specific protein contains domains related to chromatin-mediated gene expression and has been implicated in various disease states. The structure of two of the domains (PHD-bromodomain) was solved by crystallizing them as a complex with a scFv generated by phage display technology. SP140 maintains a similar overall fold to previous PHD-bromodomains and the scFv CDR loops predominately interact with the PHD, while the framework regions of the scFv makes numerous interactions with the bromodomain. Analysis of our and other complex structures suggest various protein engineering strategies that might be employed to improve the usefulness of scFvs as crystallization chaperones.

scholarly journals Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

2019 ◽  
Author(s):  
Shirafkan Kordi ◽  
Mohammad Rahmati-Yamchi ◽  
Mehdi Asghari Vostakolaei ◽  
Ali Etemadie ◽  
Abolfazl Barzegari ◽  
...  
Keyword(s):  
Phage Display ◽  
Growth Factor Receptor ◽  
Phage Display Library ◽  
Vital Role ◽  
Scfv Antibody ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Display Technology ◽  
Domain 3

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

scholarly journals Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans

2020 ◽  
Vol 21 (8) ◽  
pp. 2903
Author(s):  
Sy-Jye Leu ◽  
Yu-Ching Lee ◽  
Chi-Hsin Lee ◽  
Po-Yen Liao ◽  
Chen-Wei Chiang ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Animal Studies ◽  
New Drugs ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Candida Spp ◽  
Phage Display Technology ◽  
Multifunctional Protein ◽  
Display Technology ◽  
Opportunistic Human Pathogen

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha–enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

scholarly journals Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology

2017 ◽  
Vol 38 (6) ◽  
pp. 3915
Author(s):  
Greice Japolla ◽  
Ana Flávia Batista Penido ◽  
Greyciele Rodrigues Almeida ◽  
Luiz Artur Mendes Bataus ◽  
Jair Pereira Cunha Junior ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Bovine Herpesvirus ◽  
Single Chain ◽  
Phage Display Technology ◽  
Herpesvirus Type ◽  
Wide Range ◽  
Display Technology ◽  
Bovine Herpesvirus Type 1

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

scholarly journals Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies

2020 ◽  
Vol 21 (2) ◽  
pp. 492 ◽  
Author(s):  
Pharaoh Fellow Mwale ◽  
Chi-Hsin Lee ◽  
Liang-Tzung Lin ◽  
Sy-Jye Leu ◽  
Yun-Ju Huang ◽  
...  
Keyword(s):  
Zika Virus ◽  
Envelope Protein ◽  
Therapeutic Potential ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Virus Envelope ◽  
Phage Display Technology ◽  
High Level

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain–Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 106 and 4.5 × 106 transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV.

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