Optimization and quantification of protein synthesis inside liposomes
Optimization and quantification of protein synthesis inside liposomes
复制标题
DOI：
10.3109/08982100903402954
复制
发表时间：
2010-03-01
影响因子：
4.4
通讯作者：
Mastrobattista, Enrico
中科院分区：
文献类型：
--
作者：
Amidi, Maryam;de Raad, Markus;Mastrobattista, Enrico
合成生物学的目标是对现有的生物系统进行重新编程，或创造新的生物系统，其最终目标是获得功能可定制的人造细胞。对于后者，需要将复杂的生化反应封装到细胞大小的隔室中，如脂质体。最近，一些研究小组已经证明，通过在脂质体内包裹无细胞的蛋白质合成系统和DNA模板，可以在脂质体内重新产生感兴趣的蛋白质。虽然可以检测到，但脂质体内蛋白合成普遍较差。在这里，我们通过改变几个变量，包括脂质组成、脂质体、焦磷酸酶和T7 RNA聚合酶浓度，优化了脂质体内无细胞蛋白的合成。此外，通过使用基于活性的测定，我们量化了在优化上述变量之前和之后脂质体内DNA模板产生的全长蛋白的数量。基于模型蛋白β -半乳糖苷酶，证明了脂质体蛋白合成可以产生微克量的蛋白质（30-40 μ g/mL脂质体）。
Synthetic biology aims at reprogramming existing, or creating new, biological systems, with the ultimate aim to obtain artificial cells whose functions can be tailored. For the latter, encapsulation of complex biochemical reactions into cell-sized compartments, such as liposomes, is required. Recently, several groups have demonstrated that proteins of interest can be produced de novo within liposomes by entrapping cell-free protein-synthesis systems and DNA templates inside liposomes. Although detectable, intraliposomal protein synthesis was generally poor. Here, we have optimized intraliposomal cell-free protein synthesis by changing several variables, including lipid composition as well as liposome, pyrophosphatase, and T7 RNA polymerase concentration. Further, by using an activity-based assay, we have quantified the amount of full-length protein that was produced from DNA templates inside liposomes before and after optimization of aforementioned variables. Based on the model protein beta-galactosidase, it is demonstrated that liposomal protein synthesis can yield microgram quantities of protein (30-40 mu g/mL liposomes).