Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera
Abstract
In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.
- Authors:
-
[1];
[1];
[7];
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Univ. of Tennessee, Knoxville, TN (United States)
- ARUP Labs., Salt Lake City, UT (United States); Univ. of Tennessee, Knoxville, TN (United States)
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Specifica Inc., Santa Fe, NM (United States)
- Harvard Univ., Cambridge, MA (United States)
- Univ. of Tennessee, Knoxville, TN (United States)
- Missouri State Univ., Springfield, MO (United States)
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Specifica Inc., Santa Fe, NM (United States)
- Publication Date:
- Research Org.:
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Sponsoring Org.:
- USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE National Nuclear Security Administration (NNSA)
- OSTI Identifier:
- 1768545
- Report Number(s):
- LA-UR-20-22202
Journal ID: ISSN 1741-0126
- Grant/Contract Number:
- 89233218CNA000001; AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Protein Engineering, Design and Selection
- Additional Journal Information:
- Journal Volume: 34; Journal ID: ISSN 1741-0126
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; biological science; intrinsically fluorescent scFv antibodies; crystal structure; soluble scFv antibodies; improved expression and functionality
Citation Formats
Velappan, Nileena, Close, Devin, Hung, Li-Wei, Naranjo, Leslie, Hemez, Colin, McCullough, Donna K., DeVore, Natasha, Lillo, Antonietta Maria, and Bradbury, Andrew R. M. Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera. United States: N. p., 2021.
Web. doi:10.1093/protein/gzaa029.
Velappan, Nileena, Close, Devin, Hung, Li-Wei, Naranjo, Leslie, Hemez, Colin, McCullough, Donna K., DeVore, Natasha, Lillo, Antonietta Maria, & Bradbury, Andrew R. M. Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera. United States. https://doi.org/10.1093/protein/gzaa029
Velappan, Nileena, Close, Devin, Hung, Li-Wei, Naranjo, Leslie, Hemez, Colin, McCullough, Donna K., DeVore, Natasha, Lillo, Antonietta Maria, and Bradbury, Andrew R. M. Mon .
"Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera". United States. https://doi.org/10.1093/protein/gzaa029. https://www.osti.gov/servlets/purl/1768545.
@article{osti_1768545,
title = {Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera},
author = {Velappan, Nileena and Close, Devin and Hung, Li-Wei and Naranjo, Leslie and Hemez, Colin and McCullough, Donna K. and DeVore, Natasha and Lillo, Antonietta Maria and Bradbury, Andrew R. M.},
abstractNote = {In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.},
doi = {10.1093/protein/gzaa029},
journal = {Protein Engineering, Design and Selection},
number = ,
volume = 34,
place = {United States},
year = {Mon Feb 15 00:00:00 EST 2021},
month = {Mon Feb 15 00:00:00 EST 2021}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries
journal, March 1997
- Persic, Lidija; Roberts, Andy; Wilton, Jane
- Gene, Vol. 187, Issue 1
Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context
journal, September 2006
- Kehoe, John W.; Velappan, Nileena; Walbolt, Monica
- Molecular & Cellular Proteomics, Vol. 5, Issue 12
Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005
- Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
- Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
Recombinant Antibodies to the Small GTPase Rab6 as Conformation Sensors
journal, May 2003
- Nizak, C.
- Science, Vol. 300, Issue 5621
Quality control of antibodies for assay development
journal, September 2016
- Schumacher, Sarah; Seitz, Harald
- New Biotechnology, Vol. 33, Issue 5
Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
journal, August 1988
- Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.
- Proceedings of the National Academy of Sciences, Vol. 85, Issue 16
Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents
journal, October 2015
- Bradbury, Andrew R. M.; Plückthun, Andreas
- Protein Engineering Design and Selection, Vol. 28, Issue 10
Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen
journal, March 1992
- Hanke, T.; Szawlowski, P.; Randall, R. E.
- Journal of General Virology, Vol. 73, Issue 3
Beyond natural antibodies: the power of in vitro display technologies
journal, March 2011
- Bradbury, Andrew R. M.; Sidhu, Sachdev; Dübel, Stefan
- Nature Biotechnology, Vol. 29, Issue 3
Yeast surface display for screening combinatorial polypeptide libraries
journal, June 1997
- Boder, Eric T.; Wittrup, K. Dane
- Nature Biotechnology, Vol. 15, Issue 6
Development of Anti-Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications
journal, January 2020
- Lillo, Antonietta M.; Velappan, Nileena; Kelliher, Julia M.
- ImmunoTargets and Therapy, Vol. Volume 9
Secretion of a single-gene-encoded immunoglobulin from myeloma cells.
journal, September 1993
- Shu, L.; Qi, C. F.; Schlom, J.
- Proceedings of the National Academy of Sciences, Vol. 90, Issue 17
Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard
journal, December 2007
- Terwilliger, Thomas C.; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.
- Acta Crystallographica Section D Biological Crystallography, Vol. 64, Issue 1
Selection and characterization of FcεRI phospho-ITAM specific antibodies
journal, July 2019
- Velappan, Nileena; Mahajan, Avanika; Naranjo, Leslie
- mAbs, Vol. 11, Issue 7
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
By-passing immunization: Human antibodies from V-gene libraries displayed on phage
journal, December 1991
- Marks, James D.; Hoogenboom, Hennie R.; Bonnert, Timothy P.
- Journal of Molecular Biology, Vol. 222, Issue 3, p. 581-597
Protein production by auto-induction in high-density shaking cultures
journal, May 2005
- Studier, F. William
- Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234
Fluorescence linked immunosorbant assays using microtiter plates
journal, July 2008
- Velappan, N.; Clements, J.; Kiss, C.
- Journal of Immunological Methods, Vol. 336, Issue 2
Fluorescent Labeling of Antibody Fragments Using Split GFP
journal, October 2011
- Ferrara, Fortunato; Listwan, Pawel; Waldo, Geoffrey S.
- PLoS ONE, Vol. 6, Issue 10
A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins
journal, October 2007
- Rothbauer, Ulrich; Zolghadr, Kourosh; Muyldermans, Serge
- Molecular & Cellular Proteomics, Vol. 7, Issue 2
The use of the RACE method to clone hybridoma cDNA when V region primers fail
journal, July 1994
- Ruberti, Francesca; Cattaneo, Antonino; Bradbury, Andrew
- Journal of Immunological Methods, Vol. 173, Issue 1
Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012
- Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
- Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
High level production of soluble single chain antibodies in small-scale Escherichia coli cultures
journal, January 1997
- Kipriyanov, Sergey M.; Moldenhauer, Gerhard; Little, Melvyn
- Journal of Immunological Methods, Vol. 200, Issue 1-2
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix
journal, October 2019
- Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L.
- Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10
When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions
journal, March 2018
- Bradbury, Andrew R. M.; Trinklein, Nathan D.; Thie, Holger
- mAbs, Vol. 10, Issue 4
Leucine Zipper Dimerized Bivalent and Bispecific scFv Antibodies from a Semi-synthetic Antibody Phage Display Library
journal, March 1996
- de Kruif, John; Logtenberg, Ton
- Journal of Biological Chemistry, Vol. 271, Issue 13
Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker
journal, November 2012
- Ferrara, Fortunato; Naranjo, Leslie A.; Kumar, Sandeep
- PLoS ONE, Vol. 7, Issue 11
Specific Detection of His-Tagged Proteins with Recombinant Anti-His Tag scFv-Phosphatase or scFv-Phage Fusions
journal, January 1997
- Lindner, P.; Bauer, K.; Krebber, A.
- BioTechniques, Vol. 22, Issue 1
Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering: Extremely Stable FP Structures and Engineering
journal, May 2015
- Close, Devin W.; Paul, Craig Don; Langan, Patricia S.
- Proteins: Structure, Function, and Bioinformatics, Vol. 83, Issue 7
Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv)
journal, January 1995
- Dübel, S.; Breitling, F.; Kontermann, R.
- Journal of Immunological Methods, Vol. 178, Issue 2
Quality Issues of Research Antibodies
journal, January 2016
- Weller, Michael G.
- Analytical Chemistry Insights, Vol. 11
Selection and verification of antibodies against the cytoplasmic domain of M2 of influenza, a transmembrane protein
journal, January 2020
- Velappan, Nileena; Micheva-Viteva, Sofiya; Adikari, Samantha H.
- mAbs, Vol. 12, Issue 1
Factors Influencing the Dimer to Monomer Transition of an Antibody Single-Chain Fv Fragment †
journal, September 1998
- Arndt, Katja M.; Müller, Kristian M.; Plückthun, Andreas
- Biochemistry, Vol. 37, Issue 37
Prokaryotic expression of antibodies
journal, December 2005
- Arbabi-Ghahroudi, Mehdi; Tanha, Jamshid; MacKenzie, Roger
- Cancer and Metastasis Reviews, Vol. 24, Issue 4
Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis
journal, December 2011
- Lillo, Antonietta M.; Ayriss, Joanne E.; Shou, Yulin
- PLoS ONE, Vol. 6, Issue 12
Restricted Diversity of Antigen Binding Residues of Antibodies Revealed by Computational Alanine Scanning of 227 Antibody–Antigen Complexes
journal, November 2014
- Robin, Gautier; Sato, Yoshiteru; Desplancq, Dominique
- Journal of Molecular Biology, Vol. 426, Issue 22
Fluorescent-labeled antibodies: Balancing functionality and degree of labeling
journal, July 2010
- Vira, Shaleen; Mekhedov, Elena; Humphrey, Glen
- Analytical Biochemistry, Vol. 402, Issue 2
Green fluorescent antibodies: novel in vitro tools
journal, June 2000
- Casey, Joanne L.; Coley, Andrew M.; Tilley, Leann M.
- Protein Engineering, Design and Selection, Vol. 13, Issue 6
Assessment of the Effect of Increased Fluorophore Labelling on the Binding Ability of an Antibody
journal, April 1996
- McCormack, Teresa; O'Keeffe, Gerard; Mac Craith, Brian
- Analytical Letters, Vol. 29, Issue 6
Expression of recombinant multi-coloured fluorescent antibodies in gor -/ trxB- E. coli cytoplasm
journal, January 2011
- Markiv, Anatoliy; Beatson, Richard; Burchell, Joy
- BMC Biotechnology, Vol. 11, Issue 1
Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin
journal, December 2000
- Cloutier, S. M.; Couty, S.; Terskikh, A.
- Molecular Immunology, Vol. 37, Issue 17
High avidity scFv multimers; diabodies and triabodies
journal, December 1999
- Hudson, Peter J.; Kortt, Alexander A.
- Journal of Immunological Methods, Vol. 231, Issue 1-2
Multiplexed immuno-precipitation with 1725 commercially available antibodies to cellular proteins
journal, November 2011
- Slaastad, Heidi; Wu, Weiwei; Goullart, Luiz
- PROTEOMICS, Vol. 11, Issue 23
Module based antibody engineering: A novel synthetic REDantibody
journal, February 2011
- Markiv, Anatoliy; Anani, Bernard; Durvasula, Ravi V.
- Journal of Immunological Methods, Vol. 364, Issue 1-2
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
Choline phosphate cytidylyltransferase-α is a novel antigen detected by the anti-ERCC1 antibody 8F1 with biomarker value in patients with lung and head and neck squamous cell carcinomas: CCTα in Lung Carcinoma and HNSCC
journal, April 2014
- Vaezi, Alec E.; Bepler, Gerold; Bhagwat, Nikhil R.
- Cancer, Vol. 120, Issue 12
Exploiting recombination in single bacteria to make large phage antibody libraries
journal, January 2000
- Sblattero, Daniele; Bradbury, Andrew
- Nature Biotechnology, Vol. 18, Issue 1