An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation.
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.
Zeenko
Vladimir V
VV
Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Wang
Chuanping
C
Majumder
Mithu
M
Komar
Anton A
AA
Snider
Martin D
MD
Merrick
William C
WC
Kaufman
Randal J
RJ
Hatzoglou
Maria
M
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
D015852:Eukaryotic Initiation Factor-2; D012315:RNA Caps; D012333:RNA, Messenger; D011994:Recombinant Proteins