Human methyl-CpG binding domain protein 1, MBD domain, in complex with methylated DNA

Structure

MBD1

PDB Code

6D1T

Entry clone accession

AF078830

Entry clone source

AU94-F10

Tag

N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgrenlyfqg.

Construct sequence

mhhhhhhssgrenlyfqg

MAEDWLDCPALGPGWKRREVFRKSGATCGRSDTYYQSPTGDRIRSKVELTRYLGPACDLTLFDFKQGILCYPAPKAH

Vector

pET28-MHL

Expression host

E. coli BL21(DE3)-V2R-pRARE2

Growth medium

TB

Growth method

A fresh transformation was used to inoculate 50 mL LB media containing 50 µg/mL kanamycin and 30 µg/mL

 Chloramphenicol.  The culture was grown overnight at 37ºC with shaking. The next day this starter culture

 was used to inoculate 2 L of TB growth medium. The culture was grown in LEX at 37 ºC to OD₆₀₀ of 1.0.

IPTG-based induction was carried out according to the manufacturer’s protocol.

The temperature was reduced to 16 ºC and the culture was incubated for a further

18 hours before harvesting the cells.

Extraction buffers

Lysis buffer: 20 mM, Tris pH 7.5, 500 mM NaCl, 5% Glycerol, 0.1% NP40, 1 mM PMSF

Extraction procedure

Cells were harvested by centrifugation and pellets were stored in -80 ºC.

Prior to purification, the cell pellet was resuspended in lysis buffer.

Cells were disrupted by sonication (10 minutes) and samples were centrifuged for 60 min at 70000 g.

Purification buffers

NiNTA Elution buffer (EB): 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 250 mM Imidazole
Gel Filtration buffer: 20 mM Tris pH 7.5,150 mM NaCl, 1 mM DTT.

Purification procedure

Column 1: Affinity purification, open Ni-NTA column Procedure: The supernatant was incubated with 6mL

of 50% slurry Ni-NTA beads by rocking. After 30 min incubation at 4ºC, the beads were washed with 50 mL

of lysis buffer. The protein was eluted using ~20 mL EB.

The Column 2: Gel Filtration (Superdex S75 16/60 Hi-Load, GE Healthcare).

Then the fractions containing protein were identified on a SDS-PAGE gel.

Protein stock concentration

10 mg/ml.

Crystallization

30% PEG-550-MME, 0.1 M magnesium chloride, 0.1 M HEPES

Data collection