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Selection of affibody molecules to the ligand‐binding site of the insulin‐like growth factor‐1 receptor
Author(s) -
Li Jingjing,
Lundberg Emma,
Vernet Erik,
Larsson Barbro,
HöidénGuthenberg Ingmarie,
Gräslund Torbjörn
Publication year - 2010
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20090226
Subject(s) - receptor , biopanning , growth factor , chemistry , insulin like growth factor , ligand (biochemistry) , competitive binding , phage display , biophysics , small molecule , binding site , non competitive inhibition , flow cytometry , biochemistry , biology , peptide library , microbiology and biotechnology , peptide , peptide sequence , gene , enzyme
Affibody molecules binding to the site of hormone interaction in IGF‐1R (insulin‐like growth factor‐1 receptor) were successfully selected by phage‐display technology employing a competitive‐elution strategy during biopanning, whereby release of receptor‐bound phagemids was accomplished by competition with IGF‐1 (insulin‐like growth factor‐1). In non‐competitive selections, the elution of receptor‐bound phagemids was performed by imidazole or low‐pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA‐based assay showed that the affibody molecules generated by IGF‐1 competition during elution, in addition to affibody molecules generated in the non‐competitive selections, could compete with IGF‐1 for binding to the receptor. The affinities of the isolated variants to IGF‐1R‐overexpressing MCF‐7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z 4:40 , was shown to recognize IGF‐1R efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull‐down from cell extracts. In addition, when Z 4:40 was added to the medium of MCF‐7 cells that were dependent on IGF‐1 for efficient growth, it was found to have a dose‐dependent growth‐inhibitory effect on the cells. Applications of affibody‐based reagents for quantitative and qualitative analyses of IGF‐1R status, as well as applications of affibody‐based reagents for therapy, are discussed.