Selection of IgE-binding aptameric green fluorescent protein (Ap-GFP) by the ribosome display (RD) platform.
GFP-Ckappa fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-Ckappa fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 microg of the dual site aptameric GFP library exhibiting a diversity of 7.5x10(11) was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.
Chen
Swey-Shen
SS
The Institute of Genetics, Allergy and Immunology, 6370 Lusk Boulevard, F109-F110, San Diego, CA 92121, USA. achen.genetics.org@gmail.com
Yang
Yong-Min
YM
Barankiewicz
Teresa J
TJ
D000818:Animals; D052158:Aptamers, Peptide / Q000302:isolation & purification* / Q000378:metabolism; D015374:Biosensing Techniques; D049452:Green Fluorescent Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D051379:Mice; D009101:Multiple Myeloma / Q000276:immunology; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D052158:Aptamers, Peptide; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007073:Immunoglobulin E