ABSTRACT
The phage surface display technology, which provides a link between genetic information and gene product, is becoming an important tool in biotechnology. Cloning systems designed to express gene products on the surface of phage have proven to be useful for the display and screening of small peptides, as well as functional monomeric, homo- or heterodimeric proteins. Unfortunately, the available systems do not allow the construction of cDNA expression libraries, because translational stop codons present at the 3′ non-translated regions of eukaryotic mRNA prevent generation of fusion proteins N-terminal to phage coat proteins. Fusion to the membrane-bound C-terminus of the coat protein would hamper incorporation of the fusion product into the virion. To covalently link the expressed cDNA products to the phage surface, a strategy based on the phagemid pComb3 and the ability of the Jun and Fos leucine zippers to heterodimerize was developed. The gIII gene product was modified, allowing the production of a chimaeric protein in which the Jun leucine zipper is fused N-terminal to this viral coat protein. The modified gIII protein was then used to capture cDNA products decorated with the Fos leucine zipper, thus allowing these proteins to become structurally incorporated into phage particles during phage morphogenesis. To avoid interphage exchange of fos-cDNA fusion products, cysteines flanking both leucine zippers were engineered to provide a covalent link between the cDNA gene products and the genetic information required for their production. The resulting phagemid, pJuFo, allows expression and display of cDNA libraries on the phage surface. Screening can thus be acomplished by employing the powerful biopanning procedure, which has been very successfully used with other filamentous phage systems.
