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

Journal Article · Fri Jul 27 00:00:00 EDT 2007 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2007.05.083· OSTI ID:20991454

Yongmin, Yang ^[1]; Barankiewicz, Teresa J ^[1]; Mingyue, He ^[2]; Taussig, Michael J ^[2]; Chen, Swey-Shen ^[3]

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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)

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.

• OSTI ID: 20991454
• Journal Information: Biochemical and Biophysical Research Communications, Vol. 359, 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); ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AMPLIFICATION
ANTIBODIES
ANTIGENS
DNA
GENES
IN VITRO
NUCLEOTIDES
POLYMERASE CHAIN REACTION
PROMOTERS
RECEPTORS
SENSITIVITY
TRANSCRIPTION