Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction.
Techniques for cloning cDNAs from bacteriophage libraries immobilised on solid supports are well established. However, these techniques do not allow selective enrichment of clones expressing proteins of interest. Screening of cDNA libraries would be simplified if the proteins encoded by cDNAs could be expressed on the surface of phage. Phage carrying genes encoding proteins for which a ligand is available can be selected directly by affinity interaction [Crameri, R. & Suter, M. (1993) Gene (Amst.) 137, 69-75]. The expression products from a cDNA library from Aspergillus fumigatus have been displayed on the surface of the filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA-encoded proteins to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows screening of up to 1 x 10(10) independent clones in 50-microliters aliquots applied to a well of a microtiter plate coated with the ligand. Phage displaying IgE-binding proteins were selectively enriched 10(5)-10(6)-fold over non-specific phage after six rounds of growth and selection. The apparent molecular mass of the proteins selected from the cDNA library was in the range 20-40 kDa. Restriction enzyme analysis and preliminary sequence determination of 12 selected inserts revealed different sequences. The ability of the proteins to bind to human serum IgE was corroborated by enzyme-linked immunosorbent assay and by Western-blot analysis. The developed cloning strategy allows isolation of cDNAs encoding proteins for which a ligand is available and circumvents immobilisation of the libraries on solid-phase supports which hamper selective enrichment of clones expressing the desired protein.
Swiss Institute of Allergy and Asthma Research, Davos.
R
R
Crameri
R
R
Jaussi
G
G
Menz
K
K
Blaser
D001232:Aspergillus fumigatus / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005800:Genes, Fungal; D005814:Genes, Viral; D005821:Genetic Techniques; D015698:Genomic Library; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D005656:Fungal Proteins; D008024:Ligands; D014764:Viral Proteins; D007073:Immunoglobulin E