| The focus of my dissertation research is protein engineering. It started with modifying a well-known enzyme, bovine pancreatic phopholipase A2 (PLA2), in order to study its structure-function relationship, and advanced to the rational design and construction of Splase, a artificial restriction endonuclease with potential "anti-HIV" activity, for AIDS gene therapy. In the first part of this dissertation, extensive structural and functional studies on PLA2 have been described. These studies include the characterization of the substrate binding site residue Phe-5 and the C-terminal 9 residues. A wide range of state-of-the-art methodologies have been employed for these studies such as site-directed mutagenesis and many other molecular biology techniques, multi-dimensional Nuclear Magnetic Resonance (NMR), and computer modeling. Research related to the unnatural amino acid site-directed mutagenesis (UAA-SDM) are described in the second part of this dissertation. To facilitate the UAA-SDM, a new expression vector pUK has been constructed allowing easy purification of the high quality DNA required for UAA-SDM. We have incorporated into PLA2 an unnatural amino acid, pipecolic acid. Since the ultimate goal of protein engineering is to design and make enzymes with desired characteristics, in the third part of this dissertation, the engineering of a novel restriction endonuclease, Splase, potentially useful in AIDS gene therapy has been reported. Splase, designed to cut the HIV genome at the Sp1 sites in the long terminal repeats, was constructed by joining the DNA cleavage domain of the restriction endonuclease FokI with the zinc-finger DNA-binding domain of the transcription factor Sp1. It was shown to cleave plasmid DNAs carrying Sp1 sites, and the long terminal repeat sequence of HIV specifically near Sp1 sites. |
