Structure

Human methyltransferase dimer N6AMT1-TRMT112, methyltransferase domain, in complex with S-adenosyl-homocysteine

PDB Code

6PED

Entry clone accession

Entry clone source

SGC clone accession

Tag

N-terminal tag

Construct sequence

MGSSHHHHHHSSGLVPRGSMAGENFATPFHGHVGRGAFSDVYEPAEDTFLLLDALEAAAAELAGVEICLEVGSGS

GVVSAFLASMIGPQALYMCTDINPEAAACTLETARCNKVHIQPVITDLVKGLLPRLTEKVDLLVFNPPYVVTPPQEV

GSHGIEAAWAGGRNGREVMDRFFPLVPDLLSPRGLFYLVTIKENNPEEILKIMKTKGLQGTTALSRQAGQETLSVLKFTKS

MHHHHHHSSGRENLYFQGMKLLTHNLLSSHVRGVGSRGFPLRLQATEVRICPVEFNPNFVARMIPKVEWSAFLEAA

DNLRLIQVPKGPVEGYEENEEFLRTMHHLLLEVEVIEGTLQCPESGRMFPISRGIPNMLLSEEETES

Vector

N6AMT1 was cloned into pET28a-LIC, TRMT112 was cloned into pET15a-MHL vector, respectively.

Expression host

BL21 (DE3) Codon plus RIL (Stratagene)

Growth method

The two proteins were co-expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin. Cell were grown at 37 ºC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.2 mM, and incubated overnight at 16 ºC. Cell pellets collected by centrifugation and frozen at -80 ºC.

Extraction buffers

Lysis buffer: 20 mM Tris-HCl pH 7.5, 400 mM NaCl, 5% glycerol and 2 mM beta-mercaptoethanol

Extraction procedure

Frozen cell pellet was thawed and suspended in lysis buffer. The cells were lysed by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 5 sec pulse at half-maximal frequency (5.0), 7 second rest, for 10 minutes total sonication time per pellet. The lysate was centrifuged at 15000rpm for 1h.

Purification buffers

Wash buffer: 20 mM Tris pH 7.5, 400 mM NaCl, 5% glycerol and 25 mM imidazole;
Elution buffer: 20 mM Tris pH 7.5, 400 mM NaCl, 5% glycerol and 300 mM imidazole;
Gel filtration buffer: 20 mM Tris-HCl pH 7.5, 100 mM NaCl and 1 mM DTT.

Purification procedure

The proteins were purified by Ni-NTA agarose column and further purified by gel filtration Superdex 200 10/300 (GE Healthcare). The gel filtration buffer contains 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1 mM DTT.

Protein stock concentration

The purified protein was concentrated to 10 mg mL-1.

Crystallization

The complex was crystallized using sitting drop vapor diffusion method by mixing 1 mL protein with 1 mL reservoir solution. The crystals were obtained in 2M Na/KPO4, 7.0. The crystals were cryo-protected in the reservoir solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen.