In vitro alpha-complementation of beta-galactosidase on a bacteriophage surface.
Surface display of large multimeric non-secreted proteins is advantageous on the bacteriophage lambda compared with the widely used filamentous phage systems. A model system, the alpha-complementation of beta-galactosidase, was used for both further general characterization of protein-protein interactions on the lambda tail tube surface and for specifically probing the structure of the phage-displayed beta-galactosidase tetramer. In this complementation system, dimeric enzymatically inactive N-terminal deletion mutants of beta-galactosidase (alpha-acceptors) interact with peptides whose sequences span the region of the deletion (alpha-peptides) with the subsequent formation of tetramers and restoration of activity. The lambda phage could tolerate incorporation into their tail tubes of a limited number of copies of V protein (gpV) subunits C-terminally modified with an active alpha-peptide. Purified alpha-peptide phage showed specific in vitro alpha-complementation with an alpha-acceptor extract; the features of this reaction suggested that each complemented monomer can directly associate with an alpha-peptide displayed within the same tail tube structure. In contrast to the alpha-peptide, attempts to surface display an alpha-acceptor protein in a similar manner were unsuccessful. The implications of this work for surface-display cDNA libraries are discussed.
Dunn
I S
IS
Queensland Cancer Fund Research Unit, Department of Pathology, Medical School, University of Queensland, Brisbane, Australia. IanD@mail.kids.usyd.edu.au
D010582:Bacteriophage lambda / Q000235:genetics*; D005816:Genetic Complementation Test; D007700:Kinetics; D046911:Macromolecular Substances; D019151:Peptide Library; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013499:Surface Properties; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics*
D046911:Macromolecular Substances; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase