Method For Studying Binding Properties Of A Protein-protein Interaction Domain PDZ

A novel method for making random peptide library with genomic DNA was introduced in the first part of this dissertation. It is generally believed that genomic DNA cannot be directly used for expression of biological meaningful proteins or peptides. However, if we are to make random peptide library, genomic DNA can be considered a useful DNA fragment sources. Large genomic DNAs can be digested to short fragments and those short fragments of DNA can be considered nearly random and were used here for making random peptide library. For example, the human genomic DNA is 2.91× 10~9bp and can produce 10~7(2.91 × 10~9/256) fragments after it is fully digested with restriction endonuclease DpnII or Tsp509I, which recognizes 4 nucleotides. These fragments, which are 256bp long in average and can be cloned into any expression vectors, for example yeast two-hybrid vector pGADT7, predigested by BamHI or EcoRI, which generates compatible cohesive ends with DpnII or Tsp509I respectively. In those libraries, average fragment codes 85(256/3) amino acids. Since there are 3 stop codons in every 64 codons, the peptides expression stop at average length of 21 amino acid residues.In the first part of the dissertation, two such random peptide libraries were constructed, one was human genomic DNA digested by DpnII, in which 1.1 × 10~6 independent clones were obtained while the other was digested by Tsp509I, in which 1.4 ×10~7 independent clones were obtained.