Generation of Synthetic Antibody Fragments to Detergent Solubilized Membrane Proteins

2017 ◽  
pp. 231-243
Author(s):  
Serdar Uysal ◽  
Anthony Kossiakoff
Keyword(s):  
Membrane Proteins ◽  
Antibody Fragments ◽  
Synthetic Antibody

scholarly journals Exploring cellular biochemistry with nanobodies

2020 ◽  
Vol 295 (45) ◽  
pp. 15307-15327 ◽  
Author(s):  
Ross W. Cheloha ◽  
Thibault J. Harmand ◽  
Charlotte Wijne ◽  
Thomas U. Schwartz ◽  
Hidde L. Ploegh
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Heavy Chain ◽  
Antibody Fragments ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Chemical Functionalization ◽  
Domain Antibody ◽  
Cellular Biochemistry ◽  
Reducing Environment

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain–only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

scholarly journals CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Abdellali Kelil ◽  
Eugenio Gallo ◽  
Sunandan Banerjee ◽  
Jarrett J. Adams ◽  
Sachdev S. Sidhu
Keyword(s):  
Next Generation Sequencing ◽  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Cost Effective ◽  
Integral Membrane Proteins ◽  
Sequencing Error ◽  
Next Generation ◽  
Synthetic Antibody ◽  
Generation Sequencing

AbstractSynthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

scholarly journals The Use of Engineered Antibody Fragments to Promote Crystallization of Membrane Proteins

2014 ◽  
Vol 106 (2) ◽  
pp. 46a
Author(s):  
Sibel Kalyoncu ◽  
Jennifer L. Johnson ◽  
David P. Heaner ◽  
Ivan A. Morales ◽  
Jeongmin Hyun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Antibody Fragments

scholarly journals CellectSeq: In Silico Discovery of Antibodies Targeting Integral Membrane Proteins Combining In Situ Selections of Phage Displayed Synthetic Antibodies and Next-Generation Sequencing

2020 ◽  
Author(s):  
Abdellali Kelil ◽  
Eugenio Gallo ◽  
Jarrett Adams ◽  
Jason Moffat ◽  
Sachdev Sidhu
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Cost Effective ◽  
Integral Membrane Proteins ◽  
Native Form ◽  
Surface Receptors ◽  
Synthetic Antibody ◽  
Ngs Data ◽  
Generation Sequencing

Abstract Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal proteomic tools against human antigens. Yet, they typically depend on purified proteins, which exclude from interrogation integral membrane proteins that require the lipid bilayers to support their native form or function. Here, we present a novel Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins presented on native cells in complex environment. As proof of concept, we targeted the challenging tetraspanin receptor CD151, a target linked to cancer. First, we optimized in situ cell-based selections to enrich Ab pools for antigen-specific binders. Then, we designed novel NGS procedures to explore Ab pools diversities and abundances with enhanced accuracies. Finally, we developed novel motif-based scoring and error filtering algorithms for the comprehensive interrogation of NGS data to identify Abs with high diversities and specificities, even at extremely low abundances. We identified highly selective and diversified Abs against CD151 with abundance as low as 0.00009% for which manual sampling or identification using Abs abundances in NGS data would have been impossible. Here we show that CellectSeq enables the rapid discovery of diversified and selective antibodies against CD151, with implications for other integral membrane proteins and cell-surface receptors.

scholarly journals High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Shin-Chen Hou ◽  
Hong-Sen Chen ◽  
Hung-Wei Lin ◽  
Wei-Ting Chao ◽  
Yao-Sheng Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
Antibody Fragments ◽  
Synthetic Antibody ◽  
Antibody Libraries

scholarly journals Analysis of Assembly of Synthetic Antibody Fragments: Expression of Functional scFv with Predefined Specificity

BioTechniques ◽  
1997 ◽  
Vol 23 (3) ◽  
pp. 500-503 ◽  
Author(s):  
Norihiro Kobayashi ◽  
Eskil Söderlind ◽  
Carl A.K. Borrebaeck
Keyword(s):  
Antibody Fragments ◽  
Synthetic Antibody
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