Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

BMC Biotechnology

Research Article

Anam Qudrat¹  Kevin Truong² 

[1] Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street Room 407, Rosebrugh Building, M5S 3G9, Toronto, ON, Canada;Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street Room 407, Rosebrugh Building, M5S 3G9, Toronto, ON, Canada;Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, M5S 3G4, Toronto, ON, Canada;

关键词: Transmembrane proteins;    Modular assembly;    Endoplasmic reticulum;    Plasma membrane;    Protein engineering;    Synthetic biology;   

DOI  :  10.1186/s12896-016-0320-7

 received in 2016-06-11, accepted in 2016-12-07,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundTo achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells.ResultsWhile a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca2+ signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions.ConclusionsThis work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions.

【 授权许可】

CC BY   
© The Author(s). 2017

【 图 表 】

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