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. 2020 Aug 3;9:e59151. doi: 10.7554/eLife.59151

Figure 3. Semisynthesis strategy to generate segmentally 15N, 13C, 2H isotopically labeled Akt containing distinct C-tail phospho forms.

(A) Schematic representation of the semisynthesis strategy. (B) Alignment of Akt1 (aa 118–127) and Akt2 (aa 119–129) linkers with Akt1 Ser122 (ligation site, mutated to Cys in our study) and Akt2 Cys124 highlighted (red). (C–D) SDS-PAGE analyses of EPL reaction between S122C, Δ121, pThr308, pSer473 Akt fragment and isotopically labeled PH thioester fragment (C); and segmentally isotopically labeled pThr308, pSer473 full-length Akt purified from (C) using size exclusion chromatography (D), lanes 2–4: purified full-length Akt diluted 10-fold from stock, loaded volumes are 2.5, 5 and 10 µl, respectively, lanes 5–9: BSA standards 0.25, 0.5, 1, 2, 4 µg, dashed line: deletion of unrelated samples. M: protein markers (kDa).

Figure 3.

Figure 3—figure supplement 1. Generation of segmentally 15N, 13C, 2H isotopically labeled Akt proteins.

Figure 3—figure supplement 1.

(A) 15N, 13C, 2H isotopically labeled PH domain purified from 1 L of E. coli expression, diluted 20-fold, and loaded 5 and 10 µl (lanes 1–2); lanes: 3–7. (B–C) Pure segmentally isotopically labeled full-length pThr308 Akt proteins with non-p C-tail (B, lanes 1–3: 2.5, 5, 10 µl) and di-pSer477/pThr479 (C, lanes 1–2: 5, 10 µl) diluted 10-fold; lanes 4–7: BSA standards. (D) Steady-state kinetic plots v/[E] versus [ATP] with 20 µM GSK3 peptide for semisynthetic pThr308, pSer473 Akt proteins from two-piece (blue) and three-piece (magenta) expressed protein ligation strategies, n = 2. Note that, Akt protein obtained from three-piece ligation is lacking the N-terminal tags: Flag, HA and 6xHis. The obtained catalytic efficiencies (apparent kcat/Km values) expressed in the table (right) from at least two independent repeats performed for each assay, S.D. shown.