Human methyl-CpG binding protein 2, MBD domain, in complex with A(5CM)AC DNA

Structure

MeCP2

PDB Code

6OGK

Entry clone accession

BC011612.1

Entry clone source

AU51-B9

Tag

C-terminal hexahistidine tag: AAHHHHHH

Construct sequence

ASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELI

AYFEKVGDTSLDPNDFDFTVTGRGSPSRAAHHHHHH

Vector

pNIC-CH

Expression host

Escherichia coli BL21 (DE3)-RIL strain

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

IPTG-based induction was carried out according to the manufacturer’s protocol (final concentration for IPTG

 is 0.35 mM). The temperature was reduced to 16ºC and the culture was incubated for a further 18 hours

before harvesting the cells.

Extraction buffers

Lysis buffer: 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5 mM PMSF and 5% glycerol

Extraction procedure

Cells were harvested by centrifugation and pellets were stored in -80ºC. 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 and 300 mM imidazole
Gel Filtration buffer: 20 mM Tris-HCl, pH 7.5 and 150 mM NaCl, 1mM DTT.

Purification procedure

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

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

washed with 50 mL of lysis buffer with additional 10 mM imidazole. 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

10mg/ml.

Crystallization

50% precipitant mix 4, 0.1M buffer system 1, 0.06M divalents (morpheus A4) (from screen MD1-46)

Data collection