Murtas Giovanni, Kuruma Yutetsu, Bianchini Paolo, Diaspro Alberto, Luisi Pier Luigi
Centro Studi e Ricerche E. Fermi, Compendio Viminale, 00184 Rome, Italy.
Biochem Biophys Res Commun. 2007 Nov 9;363(1):12-7. doi: 10.1016/j.bbrc.2007.07.201. Epub 2007 Aug 15.
In a significant step towards the construction of the semi-synthetic minimal cell, a protein expression system with a minimal set of pure and specific enzymes is required. A novel cell-free transcription and translation system named PURESYSTEM (PS), consisting of a specified set of 36 enzymes and ribosomes, has been entrapped in POPC liposomes for protein synthesis. The PS has been used to transcribe and translate an Enhanced Green Fluorescent Protein (EGFP) gene from plasmid DNA. The synthesis is confirmed by the EGFP fluorescence emitting liposomes on fluorometric analysis and on confocal microscopy analysis. Furthermore the PS encapsulated into POPC liposomes can drive the expression of the plsB and plsC genes encoding for the sn-glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) involved in the first step of the "salvage pathway" for synthesis of POPC. The expression of GPAT and LPAAT in liposomes would in principle allow the production of the cell boundary from within.
在迈向构建半合成最小细胞的重要一步中，需要一个具有最少数量的纯的和特定酶的蛋白质表达系统。一种名为PURESYSTEM（PS）的新型无细胞转录和翻译系统，由一组特定的36种酶和核糖体组成，已被包裹在POPC脂质体中用于蛋白质合成。PS已被用于从质粒DNA转录和翻译增强型绿色荧光蛋白（EGFP）基因。通过荧光分析和共聚焦显微镜分析，EGFP荧光发射脂质体证实了该合成过程。此外，封装在POPC脂质体中的PS可以驱动编码参与POPC合成“补救途径”第一步的sn-甘油-3-磷酸酰基转移酶（GPAT）和1-酰基-sn-甘油-3-磷酸酰基转移酶（LPAAT）的plsB和plsC基因的表达。脂质体中GPAT和LPAAT的表达原则上允许从内部产生细胞边界。
