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In?vitro synthesis of the human calcium transporter Letm1 within cell-sized liposomes and investigation of its lipid dependency.

J. Biosci. Bioeng.. 2019; 
OkamuraKosuke,MatsushitaShuhei,KatoYasuhiko,WatanabeHajime,MatsuiAiko,OkaToshihiko,MatsuuraTom
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Gene Synthesis DNA sequence encoding human derived organic anion transporter 1 (OAT1 (NCBI reference sequence, NP_695008.1)) was synthesized (Genscript Japan, Tokyo, Japan) and cloned the into pET-myc-Letm1 vector by PCR and an In-Fusion HD kit according to the manufacturer's instructions. Get A Quote

摘要

The human mitochondrion-derived calcium transporter Letm1 was synthesized by reconstituted in?vitro transcription-translation (IVTT) in cell-sized liposomes and the dependency of Letm1 on phospholipid composition was investigated. Components for IVTT were encapsulated into cell-sized vesicles together with the DNA encoding Letm1, thereby preparing proteoliposomes. The synthesis of Letm1 and pH-dependent calcium transport activity were confirmed by flow cytometry. Finally, we investigated the effect of phospholipid composition on Letm1 transport activity and found that cardiolipin present in the mitochondrial membrane plays an important role on the transport activity of Letm1.

关键词

Cardiolipin,Human calcium transporter,In?vitro transcription-translation system,Lipid composition,Mitochondria,Proteolipo