title: An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation.
abstract: In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.
authors.affiliation: Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Zeenko
authors.forename: Chuanping
authors.initials: C
authors.lastname: Wang
authors.forename: Mithu
authors.initials: M
authors.lastname: Majumder
authors.forename: Anton A
authors.initials: AA
authors.lastname: Komar
authors.forename: Martin D
authors.initials: MD
authors.lastname: Snider
authors.forename: William C
authors.initials: WC
authors.lastname: Merrick
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
authors.forename: Maria
authors.initials: M
authors.lastname: Hatzoglou
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015852:Eukaryotic Initiation Factor-2; D012315:RNA Caps; D012333:RNA, Messenger; D011994:Recombinant Proteins