RRC ID 44112
Author Masutani M, Machida K, Kobayashi T, Yokoyama S, Imataka H.
Title Reconstitution of eukaryotic translation initiation factor 3 by co-expression of the subunits in a human cell-derived in vitro protein synthesis system.
Journal Protein Expr Purif
Abstract 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.
Volume 87(1)
Pages 5-10
Published 2013-1-1
DOI 10.1016/j.pep.2012.10.001
PII S1046-5928(12)00267-7
PMID 23063735
MeSH Cell-Free System Chromatography, Affinity Eukaryotic Initiation Factor-3 / biosynthesis* Eukaryotic Initiation Factor-3 / genetics Eukaryotic Initiation Factor-3 / isolation & purification HeLa Cells Humans Protein Biosynthesis* Protein Subunits / biosynthesis* Protein Subunits / genetics Protein Subunits / isolation & purification Sequence Deletion Transcription, Genetic
IF 1.513
Times Cited 7
Human and Animal Cells HeLa S3