DISSECTION OF NRP,A PEPTIDE SYNTHETASE REQUIRED FOR MYCOBACTERIUM TUBERCULOSIS VIRULENCE

KIMBERLY M. RAMOS, JULIAN A. FERRERAS, FEDERICO A. DI LELLO, PATRICIA FLORIT, LUIS E.N. QUADRI

Austin, Texas, Estados Unidos

Conferencia; SACNAS 2004 National Conference; 2004

Society for Advancement of Chicanos and Native Americans in Science

The product of gene rv0101 is believed to be essential to the virulence of Mycobacterium tuberculosis.This gene probably codes for Nrp, a non-ribosomal peptide synthetase (NRPS).The domains of Nrp were predicted by comparing its sequence to other proteins in the NRPS family.Theoretical condensation, adenylation, peptidyl carrier, and reductasedomains were assigned. Genes rv0099 and rv0100 are believed to code for proteins that participate together with Nrp in the production of a lipopeptide vital for virulence. In order to prove that these domains are functional as predicted, each domain must be expressed, purified, and evaluated for activity in vitro. Through these experiments, information pertaining to the nature of the product of this Nrp can be obtained. Because the Nrp product is necessary for the virulence of M. tuberculosis, Nrp is a promising antibiotic target. In order to purify the domains, each gene fragment (coding for one or more domains) was individually cloned into the plasmid TOPO and transformed into DH5a cells.The fragments were each subcloned into the plasmid pSMT3 and transformed into BL21 cells for protein expression.The proteins, once produced, were purified from cell lysate using a Nickel column to which the His tag of each construct bound. Expressionand purification for rv0099, rv0100, adenylation domains 1 and 2 (A1, A2), condensation domains 1 and 2, peptidyl carrier protein 1, and reductase domain were optimized. Attempts were made to verify activity of A1 and A2 and to determine the cognate amino acid of each domain.