Intein-mediated labeling of HP1α

Human HP1α was expressed in E. coli as a C-terminal fusion to the N-terminal half of the Npu split intein, followed by a hexahistidine tag. After purification by Ni-affinity and anion-exchange chromatography (to remove residual DNA), HP1α-NpuN was bound to a column of resin-linked NpuC. A Thz₁-G₂-C₃-CONH₂ tripeptide (P1) was synthesized by FMOC solid-phase peptide synthesis (Thz stands for thiazolidine). The peptide was subsequently reacted with Atto532-iodoacetamide in 20 mM Tris buffer, pH 7.5, followed by opening of Thz by incubation with 500 mM methoxylamine at pH 5. 1 mM purified peptide was then added to the column-bound HP1α in in situ EPL buffer (50 mM MPAA, 200 mM MESNA, 100 mM phosphate, 10 mM TCEP, 1 mM EDTA, pH 7.8). After overnight incubation at RT, the ligated protein was eluted, purified by size-exclusion chromatography, mixed with glycerol to 30% and flash frozen in small aliquots for later use.