Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
(a) The genes plsB and plsC coding for the GPAT and LPAAT enzymes, respectively, were expressed by in vitro transcription translation (IVTT) in the presence of SUVs. Spontaneously assembled proteoliposomes containing synthesized GPAT and LPAAT proteins were isolated by ultracentrifugation (floatation method) and the protein content was analysed by SDS-PAGE. Activity assays were performed by adding the phospholipid precursors G3P and acyl-CoA (shown in the reaction scheme is palmitoyl-CoA, p-CoA) either before or subsequent to IVTT reaction. Biosynthesis of 1,2-diacylglycerol-3-phosphate (here DPPA) occurs in a two-step acyl transfer reaction catalysed by the GPAT and LPAAT enzymes. The intermediate product 1-acylglycerol-3-phosphate (here 16:0 LPA) and two free CoA molecules are also formed. After reaction the lipid fraction was extracted and assayed by LC-MS. To quantify the enrichment of vesicles with synthesized lipids, liposomes were purified by immobilization on beads before the lipid extraction step. (b) Cell-free expression of either the plsB or plsC gene (no gene as negative control) occurred for 3 h at 37°C in the presence of 100-nm SUVs and of GreenLys reagent (tRNA-loaded fluorescent amino acid) for fluorescence labelling of translation products. Reconstituted proteoliposomes were purified and membrane-integrated proteins separated by SDS-PAGE were visualized with coomassie brilliant blue (CBB) staining and fluorescence scanning. As shown with CBB staining the PURE system background proteins (lane 7) can efficiently be eliminated by purification, while the GPAT and LPAAT protein bands were co-purified with the SUVs (lanes 9,10). Isolation of acyltransferase enzymes is also visible on the fluorescence scan (lanes 4,5). The lower bands on lanes 2,3 correspond to background signal from the GreenLys reagent. (c) Normalized chromatogram of lipids as measured by LC-MS operating in multiple reaction monitoring (MRM) mode with negative polarity. In this example, 16:0 LPA, DOPG, DPPA, DOPE and cardiolipin were clearly resolved. Since the quaternary amine has a permanent positive charge, DOPC is not well detected in the negative mode. It was also possible to detect 18:1 LPA, DOPA, DPPS, DPPG and DPPE (Figure A in S1 File).