Femtoliter compartment in liposomes for in vitro selection of proteins.
The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.
Sunami
Takeshi
T
Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka 565-0871, Japan.
Sato
Kanetomo
K
Matsuura
Tomoaki
T
Tsukada
Koji
K
Urabe
Itaru
I
Yomo
Tetsuya
T
D002451:Cell Compartmentation; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D018716:Molecular Mimicry; D014176:Protein Biosynthesis / Q000235:genetics; D011506:Proteins / Q000032:analysis / Q000378:metabolism*
D008081:Liposomes; D011506:Proteins; D049452:Green Fluorescent Proteins