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Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

Abstract

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.
Authors:
Yongmin, Yang; [1]  IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)]; Barankiewicz, Teresa J; [1]  IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)]; Mingyue, He; [2]  Taussig, Michael J; [2]  Chen, Swey-Shen [3] 
  1. Institute of Genetics, San Diego, CA 92121-2233 (United States)
  2. Babraham Institute, Cambridge CB2 4AT (United Kingdom)
  3. Institute of Genetics, San Diego, CA 92121-2233 (United States) and IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)
Publication Date:
Jul 27, 2007
Product Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 359; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2007.05.083; PII: S0006-291X(07)01029-7; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Subject:
60 APPLIED LIFE SCIENCES; AMPLIFICATION; ANTIBODIES; ANTIGENS; DNA; GENES; IN VITRO; NUCLEOTIDES; POLYMERASE CHAIN REACTION; PROMOTERS; RECEPTORS; SENSITIVITY; TRANSCRIPTION
OSTI ID:
20991454
Country of Origin:
United States
Language:
English
Other Identifying Numbers:
Journal ID: ISSN 0006-291X; BBRCA9; TRN: US07R2249014843
Submitting Site:
INIS
Size:
page(s) 251-257
Announcement Date:
Mar 26, 2008

Citation Formats

Yongmin, Yang, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Barankiewicz, Teresa J, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Mingyue, He, Taussig, Michael J, and Chen, Swey-Shen. Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.05.083.
Yongmin, Yang, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Barankiewicz, Teresa J, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Mingyue, He, Taussig, Michael J, & Chen, Swey-Shen. Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display. United States. doi:10.1016/j.bbrc.2007.05.083.
Yongmin, Yang, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Barankiewicz, Teresa J, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Mingyue, He, Taussig, Michael J, and Chen, Swey-Shen. 2007. "Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display." United States. doi:10.1016/j.bbrc.2007.05.083. https://www.osti.gov/servlets/purl/10.1016/j.bbrc.2007.05.083.
@misc{etde_20991454,
title = {Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display}
author = {Yongmin, Yang, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Barankiewicz, Teresa J, IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)], Mingyue, He, Taussig, Michael J, and Chen, Swey-Shen}
abstractNote = {Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.}
doi = {10.1016/j.bbrc.2007.05.083}
journal = {Biochemical and Biophysical Research Communications}
issue = {2}
volume = {359}
place = {United States}
year = {2007}
month = {Jul}
}