Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
Fig 2
Two-step acyl transfer reaction mediated by cell-free synthesized GPAT and LPAAT enzymes.
(a) LC-MS analysis of the GPAT and LPAT reaction products. The lipid precursors G3P and palmitoyl-CoA (p-CoA), or p-CoA and 16:0 LPA (66.6 μM each, except in two-enzyme cascade experiments, where p-CoA concentration was 133.3 μM) were added after the IVTT reactions performed in the presence of SUVs. The two enzymes were assayed separately in their respective activity buffer or together in the reducing buffer known to support GPAT activity. Negative controls in GPAT and LPAAT activity buffers were performed using the DHFR and LacI genes. For combined GPAT and LPAAT reactions, controls were conducted without G3P. Error bars in single-enzyme experiments are s.e.m. from multiple measurements of one sample. In the GPAT and LPAAT co-expression experiments data are mean and s.e.m. across four independent samples; For each repeat the sample was injected multiple times, the average value of the different injections was calculated and data are reported as the mean and standard error of independent trials. Student t-test analysis: *P<0.015, **P<0.025. (b) Acyltransferase activity as measured using a fluorescence-based assay in which released CoA reacts with a fluorogenic substrate. Negative controls for GPAT and LPAAT activity were performed using the DFHR and LacI genes, respectively. DTT was dialysed out after the IVTT reaction to create the non-reducing conditions compatible with the assay. Blank was measured from the buffer included in the fluorescence-based CoA assay kit. Data are mean values and s.e.m. of two independent experiments. Student t-test analysis: *Difference statistically not significant, **P<0.23.