In Vitro Synthesis of DYRK1A Kinase

The PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Beverley, MA, USA), which is a reconstituted E. coli-based in vitro transcription-translation system, was used to express a DYRK1A construct comprising the catalytic domain of DYRK1A fused to an N-terminal Strep-tag II sequence. Reactions were run in a total volume of 10 μL with 10 ng/μL pET-ST2-DYRK1A [9 (link)] and 80 ng/μL SF3B1-NT-His6 as a substrate [51 (link)] at 37°C for 1 h. AnnH75 or solvent control (3% DMSO) was added as indicated. Reactions were stopped by adding 2x Laemmli sample buffer and 5 mM EDTA. Tyrosine autophosphorylation of DYRK1A and phosphorylation of SF3B1 on Thr434 was analysed by western blotting. Band intensities were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany).