Project 3 - Molecular Evolution of Human PON to Design Enhanced Catalytic Efficiency for Hydrolysis of Nerve Agents; Annual rept. 29 Jan 2007-28 Jan 2008

摘要

The long-term objective of this effort is to develop a generic gene shuffling-based technology to rapidly screen libraries of 1010 proteins/peptides encoded by DNA libraries for identifying biomolecules that can intercept both existing and emerging organophosphate-based chemical warfare nerve agents (CWNA). The specific milestones for year 1 were (a) Proof of concept for the proposed technology; (b) Generation of 4-6 libraries based on recombinant PON1 and PON3 based on gene shuffling and random mutations (c) synthesis of OP model compounds. In general all 1st year milestones were met and well beyond specifically (i) the generation of recombinant PON3 variants and (ii) generation of recombinant PON1 variants two of them (3B3 and 2B4) were overexpressed purified and found to have a 72- and 13-fold increased kcat/Km relative to the wild-type like enzyme toward a cyclosarin model compound. Notably the increased activity of these variants seems to be highly selective e.g. the activity of the two variants with the diethyl phosphoryl analog DEPCyC shows one to be lower and the other to be only 3-fold higher than the wild-type PON1. Relevance: this technology is envisaged to provide rapid discovery of pretreatment and post challenge therapeutic drugs against existing and emerging CWNA threats and will shorten the time from emergence of a threat to identification of potential counter-measures to a few days or weeks.