Reconstitution of eukaryotic translation initiation factor 3 by co-expression of the subunits in a human cell-derived in vitro protein synthesis system.
Many biologically important factors are composed of multiple subunits. To study the structure and function of the protein complexes and the role of each subunit, a rapid and efficient method to prepare recombinant protein complexes is needed. In this work, we established an in vitro reconstitution system of eukaryotic translation initiation factor (eIF) 3, a protein complex consisting of 11 distinct subunits. A HeLa cell-derived in vitro coupled transcription/translation system was programmed with multiple plasmids encoding the 11 eIF3 subunits in total. After incubation for several hours, the eIF3 complex was purified through tag-dependent affinity chromatography. When eIF3l, one of the nonessential subunits of eIF3, was not expressed, the eIF3 complex that was devoid of eIF3l was still obtained. Both the 11 subunits complex and the eIF3l-less complex were as active as native eIF3 as observed by a reconstituted translation initiation assay system. In conclusion, the cell-free co-expression system should be a feasible and rapid system to reconstitute protein complexes.
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2280, Japan.
Mamiko
M
Masutani
Kodai
K
Machida
Tominari
T
Kobayashi
Shigeyuki
S
Yokoyama
Hiroaki
H
Imataka
D002474:Cell-Free System; D002846:Chromatography, Affinity; D039621:Eukaryotic Initiation Factor-3 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion; D014158:Transcription, Genetic
D039621:Eukaryotic Initiation Factor-3; D021122:Protein Subunits