To obtain the selleck chem inhibitor full length sequence, specific Inhibitors,Modulators,Libraries primers based on both, globe artichoke and cardoon, partial cDNA sequences, were designed for 3 and 5 end amplification as described in Comino et al. Using ClustalW with standard parameters, the C. cardunculus full length amino acid sequences were aligned with the publicly available acyltransferases transferring hydroxycinnamoyl groups to acceptors from the shikimate pathway. Phylogenetic anal ysis was conducted using MEGA version 3. 0. Heterologous expression of globe artichoke HQT in E. coli and enzymatic assays The globe artichoke HQT open reading frame was amplified using HQT For and HQT Rev primers, which contain additional restriction sites, respectively, NdeI and BamHI. In a first step the amplified fragment was digested with NdeI and partially with BamHI.
This partial second digestion being necessary because of the presence of an internal BamHI restriction site. The restricted PCR fragment Inhibitors,Modulators,Libraries was finally ligated into Inhibitors,Modulators,Libraries the clon ing site of Nde I Bam HI digested pET3a plasmid. Inhibitors,Modulators,Libraries The resulting recombinant pET3a HQT plasmid was transferred into E. coli strain BL21 pLysE, and grown on a selective medium. Individ ual colonies were transferred to 4 ml LB medium and incubated for 12 h at 37 C. Two ml of this bacterial pre culture were transferred in 50 ml LB medium and grown for 3 h at 28 C prior to an isopropyl D thiogalactopyra noside induction during 8 h at 28 C. After centrifugation for 10 min at 5000 g, the pellet was resuspended in 1 ml of phosphate buffered saline pH 7.
5 and lysed by three cycles of freezing and thawing, followed by three bursts of 30 s sonication on ice. Sonicated cells were centrifuged Inhibitors,Modulators,Libraries at 4 C and 14,000 g for 5 min, and the super natant was assayed for HQT activity, and profiled by SDS PAGE using Coomas sie brilliant blue staining. Negative controls used comparable preparations harbouring an empty vector. The recombinant proteins were used for enzyme assays. CGA was purchased from Sigma Aldrich, and quinic acid from Fluka. CoA esters were synthesised using the procedure proposed by Beuerle and Pichersky. 4CL enzyme was kindly pro vided by Dr. Douglas. The 20l reaction mixture contained 100 mM phosphate buffer, 1 mM dithiothreitol, between 50 ng and 1g of protein, and the various substrates at con centrations ranging from 0. 1 mM to 5 mM. The reverse reaction, i. e.
conversion of chlorogenic acid and CoA SH into caffeoyl CoA, was tested as follow 50 ng to 1g protein was incubated in presence of 1 mM dithioth reitol, 100M of chlorogenic acid and 100M CoA. Reac tions were incubated at 30 C for 30 min, stopped by the addition of 20l of acetonitrile HCl and products were research use analysed by reverse phase HPLC on a C18 column. The two solvents used are 90% H2O, 9. 9% CH3CN, 0. 1% HCOOH and 80% CH3CN, 19. 9% H2O, 0. 1% CH3COOH.