期刊
EUROPEAN JOURNAL OF BIOCHEMISTRY
卷 268, 期 12, 页码 3407-3415出版社
WILEY
DOI: 10.1046/j.1432-1327.2001.02240.x
关键词
phage display; epitope mapping; affinity maturation; elongation factor; subcellular localization
To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a K-d of 9 nm to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.
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