Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands.
cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody-antigen, protein- protein and DNA-protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.
Ansuini
Helenia
H
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy.
Cicchini
Carla
C
Nicosia
Alfredo
A
Tripodi
Marco
M
Cortese
Riccardo
R
Luzzago
Alessandra
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry*; D001921:Brain / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D019922:GAP-43 Protein / Q000276:immunology; D015870:Gene Expression; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D019809:Streptavidin / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; C041745:D protein, Enterobacteria phage lambda; D018076:DNA, Complementary; D019922:GAP-43 Protein; D008024:Ligands; D019151:Peptide Library; D014764:Viral Proteins; D001710:Biotin; D019809:Streptavidin