Optimization and quantification of protein synthesis inside liposomes.
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 microg/mL liposomes).
Amidi
Maryam
M
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands.
de Raad
Markus
M
de Graauw
Helma
H
van Ditmarsch
Dave
D
Hennink
Wim E
WE
Crommelin
Daan J A
DJ
Mastrobattista
Enrico
E
D001695:Biology; D002474:Cell-Free System / Q000378:metabolism; D002477:Cells / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D008081:Liposomes; D011506:Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase