Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production.
A cloning and expression system allowing display of functional cDNAs or other gene products on the surface of filamentous phage has been developed, exploiting the high-affinity interaction of the Jun and Fos leucine zippers. Gene jun was expressed from a lacZ promoter as a fusion protein with the viral coat protein, pIII, thereby being structurally incorporated into phage particles during infection with a helper phage. Using a second lacZ promoter of the phagemid, gene fos was co-expressed as an N-terminal fusion peptide to cDNA library gene products, so that the resulting Fos-fusion proteins could become associated with the Jun-decorated phage particles. To avoid interphage exchange of fos-cDNA fusion products, cysteines were engineered at the N- and C-termini of each of the leucine zippers, providing a covalent link of the cDNA gene product to the genetic instructions required for its production. Dissociation between phage and cDNA gene products was readily achieved using reducing agents. Phages displaying gene products covalently anchored on their surface via the modified leucine zippers can be selectively enriched 10(4)-10(6)-fold over nonspecific phages using antibodies. Thus, this cloning system allows rapid isolation of rare mRNA products from complex cDNA libraries by enrichment with appropriate ligands. This approach should allow the expression and cloning of dimeric proteins by cDNA shuffling.
Crameri
R
R
Swiss Institute of Allergy and Asthma Research, Davos.
Suter
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D015723:Gene Library; D016762:Genes, fos; D016758:Genes, jun; D017912:Inovirus; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins