Selection of antigenic markers on a GFP-Ckappa fusion scaffold with high sensitivity by eukaryotic ribosome display.
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 (Ckappa) 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 Ckappa (3') were selected by anti-GFP or anti-Ckappa 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 10fg of the 1kb 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.
Yang
Yong-Min
YM
The Institute of Genetics, San Diego, CA 92121-2233, USA.
Barankiewicz
Teresa J
TJ
He
Mingyue
M
Taussig
Michael J
MJ
Chen
Swey-Shen
SS
D000818:Animals; D000941:Antigens / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D002474:Cell-Free System; D005819:Genetic Markers / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008957:Models, Genetic; D019151:Peptide Library; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
D000941:Antigens; D005819:Genetic Markers; D019151:Peptide Library; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D049452:Green Fluorescent Proteins