title: Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display.
abstract: 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 Kd 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.
authors: Institute of Molecular and Structural Biology, Department of Biostructural Chemistry, University of Aarhus, Denmark. svend@cajal.mbb.ki.se
authors: S
authors: S
authors: Kjaer
authors: T
authors: T
authors: Wind
authors: P
authors: P
authors: Ravn
authors: M
authors: M
authors: Østergaard
authors: B F
authors: BF
authors: Clark
authors: A
authors: A
authors: Nissim
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002461:Cell Line, Transformed; D017931:DNA Primers; D018604:Epitope Mapping; D015977:Eukaryotic Initiation Factor-1; D005455:Fluorescent Antibody Technique; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D007135:Immunoglobulin Variable Region; D010448:Peptide Initiation Factors; C026022:eukaryotic peptide initiation factor-1A