Exploiting Molecular Diversity of Enzymes Based on Phage Display: Development of Novel Enzymatic Catalysts

摘要

Barnase was successfully displayed on the surface of m13 phage as an N-terminal fusion with coat protein III. The successful display was verified by Western Blotting of purified phage. For selection of a thermostable barnase, the buried residues Ala11, Leu14, Leu20, Ala74, Ile88, Tyr90, and Ile96 were replaced randomly with Phe, Leu, Val, Ile, Ala, Pro, Gly, Ser, or Thr, giving a library of 2x10(exp 6) members. The transformation yield obtained (4x10(exp 6)) allows sampling of all possible members. We are currently selecting the phage library on immobilized barstar (the femtomolar inhibitor of barnase) at elevated temperature. Barstar N7C, a mutant constructed in this lab, was linked via a disulfide to a chromatography support. This allows specific elution under mild reducing conditions. Barnase has also been successfully expressed on the surface of phage when attached to the C-terminus of an Fab light chain, with the heavy chain attached to the gene III protein. Again, western blotting analysis was used to verify the success of display. Replacement of the barnase gene with cDNA should allow the successful surface display of cDNA libraries on phage.