Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display.
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.
Kjaer
S
S
Institute of Molecular and Structural Biology, Department of Biostructural Chemistry, University of Aarhus, Denmark. svend@cajal.mbb.ki.se
Wind
T
T
Ravn
P
P
Østergaard
M
M
Clark
B F
BF
Nissim
A
A
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
D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D007135:Immunoglobulin Variable Region; D010448:Peptide Initiation Factors; C026022:eukaryotic peptide initiation factor-1A