期刊
ADVANCED SCIENCE
卷 5, 期 10, 页码 -出版社
WILEY
DOI: 10.1002/advs.201800524
关键词
cell-free protein synthesis; hexahistidine-fused membrane proteins; nickel-chelating liposomes; polyhistidine/nickel-chelate affinity; proteoliposomes
资金
- Exploratory Research for Advanced Technology Department of the Japan Science and Technology Agency (JST-ERATO)
- JSPS KAKENHI [JP15K21119, JP16H06313]
Integral membrane proteins (IMPs) modulate key cellular processes; their dysfunctions are closely related to disease. However, production of IMPs in active conformations for further study is hindered by aggregation and toxicity in living expression systems. IMPs are therefore produced in cell-free systems employing liposome chaperoning, but membrane integration of the nascent IMPs is suboptimal and orientation of the integrated proteins remains uncontrollable. Thus, an artificial membrane protein sorting system is developed, based on polyhistidine/nickel-chelate affinity, combined with cell-free membrane protein synthesis. Its proof of concept is demonstrated with a N-terminal hexahistadine-fused conexin-43 (NHis-Cx43) model IMP. Nickel-chelating liposomes efficiently incorporate twofold newly synthesized NHis-Cx43 compared with Cx43. NHis-Cx43, when synthesized in this system, forms dye-permeable hemichannels, similar to plasma membrane pores formed by Cx43 in cells. The topology of incorporated NHis-Cx43 indicates two orientations in the liposomal membranes. However, NHis-Cx43 orientation is controlled, resulting in single topology, by combination of the natural molecular chaperone DnaKJE. Successful synthesis and at least 4.5-fold increase lipid incorporation are also achieved with three other NHis-fused IMPs, including alpha-helix and beta-barrel IMPs. Overall, this simple membrane protein sorting system is usable combined with molecular chaperones to prepare proteoliposomes for many applications.
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