Figure 6

Characterization of the recombinant IAL in E. coli. (A) Agarose gel electrophoresis of the cDNA of the ial gene obtained by RT-PCR (RT). The 1-kb Ladder plus molecular marker (Invitrogen) is indicated as M. (B) Schematic representation of plasmid pULCT-ial. (C) SDS-PAGE showing the overexpression of the ial gene in E. coli at 37°C. M: molecular mass marker; -I: uninduced cells; 37°C: total cell extracts obtained after a 5h-induction with IPTG at 37°C; I.B.: inclussion bodies obtained after a 5h-induction with IPTG at 37°C. (D) SDS-PAGE showing the overexpression of the ial gene in E. coli at 26°C. M: molecular mass marker; -I: uninduced cells; 26°C: soluble cell extracts obtained after a 5h-induction with IPTG at 26°C. Note the lack of the 40-kDa band and the presence of the 28-kDa band. (E) Biossay carried out to determine the in vitro phenylacetyl-CoA: 6-APA acyltransferase activity (see Methods) present in the soluble extracts of E. coli overexpressing either the ial (IAL) or the penDE (IAT) genes. As a negative control, the reaction mixture was used without the addition of soluble extracts from E. coli overexpressing the penDE gene (C-).