Journal:

Article Title: Phosphorylation of eIF4E attenuates its interaction with mRNA 5? cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

doi: 10.1261/rna.2133403

Figure Lengend Snippet: Characteristic of semisynthetic eIF4Es. (A,B) Expression and ligation reaction visualized on a Coomassie-stained 12% SDS-PAGE gel. (A) (lane 1) Crude extract from uninduced cells, (lane 2) crude extract from cells induced by 0.5 mM IPTG at 37°C for 3 h, (lane 3) insoluble fraction of lysed cells, (lane 4) soluble fraction of lysed cells, (lane 5) fusion protein after refolding from inclusion bodies loaded on chitin column, (lane 6) chitin column flow through, (lane 7) chitin beads before cleavage with ethanethiol, (lane 8) protein marker weight standards (SIGMA); (B) (lane 1) chitin beads before cleavage with ethanethiol, (lane 2) chitin beads after cleavage with ethanethiol, (lane 3) product of ligation reaction of two fragments eIF4E loaded on ion-exchange column, (lane 4) flow through, (lane 5) semisynthetic phosphorylated eIF4E purified on Mono SP column, (lane 6) protein marker weight standards (SIGMA), (lane 7) recombinant murine eIF4E (28–217). (C) Characteristic of semisynthetic proteins by electrospray ionization MS. (Top) Wild-type murine eIF4E (28–217), predicted mass: 22113.0 daltons, (middle) semisynthetic unphosphorylated eIF4E (28–217)T205C, predicted mass: 22115.2 daltons, (bottom) semisynthetic phosphorylated eIF4E (28–217)T205C/S209phos, predicted mass: 22195.0 daltons.

Article Snippet: The reconstruction of eIF4E (28–204) in fusion with intein appeared to be a very efficient method to prepare milligram amounts of pure fusion protein with the active intein. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIGURE 3. caption a7 Characteristic of semisynthetic eIF4Es. ( A,B ) Expression and ligation reaction visualized on a Coomassie-stained 12% SDS-PAGE gel. ( A ) (lane 1 ) Crude extract from uninduced cells, (lane 2 ) crude extract from cells induced by 0.5 mM IPTG at 37°C for 3 h, (lane 3 ) insoluble fraction of lysed cells, (lane 4 ) soluble fraction of lysed cells, (lane 5 ) fusion protein after refolding from inclusion bodies loaded on chitin column, (lane 6 ) chitin column flow through, (lane 7 ) chitin beads before cleavage with ethanethiol, (lane 8 ) protein marker weight standards (SIGMA); ( B ) (lane 1 ) chitin beads before cleavage with ethanethiol, (lane 2 ) chitin beads after cleavage with ethanethiol, (lane 3 ) product of ligation reaction of two fragments eIF4E loaded on ion-exchange column, (lane 4 ) flow through, (lane 5 ) semisynthetic phosphorylated eIF4E purified on Mono SP column, (lane 6 ) protein marker weight standards (SIGMA), (lane 7 ) recombinant murine eIF4E (28–217). ( C ) Characteristic of semisynthetic proteins by electrospray ionization MS. ( Top ) Wild-type murine eIF4E (28–217), predicted mass: 22113.0 daltons, ( middle ) semisynthetic unphosphorylated eIF4E (28–217)T205C, predicted mass: 22115.2 daltons, ( bottom ) semisynthetic phosphorylated eIF4E (28–217)T205C/S209phos, predicted mass: 22195.0 daltons.

Techniques: Expressing, Ligation, Staining, SDS Page, Marker, Purification, Recombinant