Genes encoding ankyrin domains of KANK1 (residues 1080–1329) were
synthesized by Sangon Biotech (Shanghai) and cloned into a modified
pET28-MHL (GenBankTM accession number EF456735). Expression plasmid
was transformed into E. coli BL21 (DE3), and was overexpressed at 16
°C for 18 h in the presence of 1 mM isopropyl
1-thio- -D-galactopyranoside. ankyrin domain of KANK1 was first
purified by a fast flow nickel-nitrilotriacetic acid column(GE
Healthcare). N-terminal His6 tags of recombinant protein was removed
by tobacco etch virus protease. Then the cleaved
recombinant proteins were further purified by Superdex 75 gel
filtration and mono Q ion exchange (GE Healthcare). The purified
protein was concentrated to 15 mg/mol and stored at -80 °C.
The apo-form of the KANK1 ankyrin domain(residues 1080–1329) was
crystallized by mixing an equal volume of 15 mg/ml protein with
crystallization buffer (0.1M HEPES, pH 7.5, 0.2M lithium sulfate
monohydrate, and 25% PEG 3350).Before flash-freezing crystals in
liquid nitrogen, all crystals were soaked in a cryoprotectant
consisting of 90% reservoir solution plus 10% glycerol.
