Recombinant human ERK2/MAPK1 were produced in E. coli bacteria (BL21 strain), described as above. The cDNA encoding human ERK2 (NM_002745.4) was synthesized as a gene fragment (gBlock, Integrated DNA Technologies), cloned into pGEX-KG vector, and expressed in bacteria as GST fusion proteins with a linker peptide sequence recognized by rhinovirus 3C protease. At the end of aforementioned purification process, GST cleavage by rhinovirus 3C protease was carried out in kinase buffer: 25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2. The yield was around 2–5 mg from half a liter culture of E. coli BL21 harboring pGEX-ERK2.
