Structure
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MeCP2
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PDB Code
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6OGJ
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Entry clone accession
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BC011612.1
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Entry clone source
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AU51-B9
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Tag
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C-terminal hexahistidine tag: AAHHHHHH
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Construct sequence
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ASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYL
INPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSAHHHHHH
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Vector
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pNIC-CH
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Expression host
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Escherichia coli BL21 (DE3)-RIL strain
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Growth medium
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TB
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Growth method
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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.
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Extraction buffers
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Lysis buffer: 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5 mM PMSF and 5% glycerol
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Extraction procedure
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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.
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Purification buffers
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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.
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Purification procedure
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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.
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Protein stock concentration
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10mg/ml.
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Crystallization
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30% PEG 2000 MME, 0.2 M potassium bromide
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Data collection
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