L3MBTL

PDB:2RJF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_056293
Entry Clone Source:
SGC Clone Accession:L3MBTL1_3:A3-APC053
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgrenlyfq*g
Host:E Coli BL21(DE3) RIL CodonPlus

Construct

Prelude:
Sequence:

GEKKECWSWESYLEEQKAITAPVSLFQDSQAVTHNKNGFKLGMKLEGIDPQHPSMYFILTVAEVCGYRLRLHFDGYSECHDFWVNANSPDIHPAGWFEKTGHKLQPPKGYKEEEFSWSQYLRSTRAQAAPKHLFVSQSHSPPPLGFQVGMKLEAVDRMNPSLVCVASVTDVVDSRFLVHFDNWDDTYDYWCDPSSPYIHPVGWCQKQGKPLTPPQDYPDPDNFCWEKYLEETGASAVPTWAFKVRPPHSFLVNMKLEAVDRRNPALIRVASVEDVEDHRIKIHFDGWSHGYDFWIDADHPDIHPAGWCSKTGHPLQPPLGPRE

Vector:p28a-MHL

Growth

Medium:TB
Antibiotics:
Procedure:A glycerol stock was used to innoclate 20 mL LB media containing 50 µg/mL kanamycin. The culture was grown overnight at 37ºC with shaking. The next day this starter culture was used to innoculate 2L of TB medium which contained 50 μg/mL kanamycin. The culture was grown in LEX at 37ºC to OD600 ~1.1 and was induced with the addition of 0.5 mM IPTG. The temperature was reduced to 18ºC and the culture was incubated for a further 16 hours before harvesting the cells.

Purification

Procedure
Column 1: Affinity purification, open Ni-NTA columnProcedure: The supernatant was incubated with 6mL of 50% slurry Ni-NTA beads by rocking. After 1 hour incubation at 4ºC, the beads were washed with 50 mL of lysis buffer. The protein was eluted using ~20mL EB. Column 2: Gel filtration, HiLoad 26/60 Superdex 200 Prep GradeProcedure: The eluent from Ni column was loaded onto the gel filtration column in GF buffer at 2.5 mL/min, fraction size 4mL. The fractions containing protein were identified on a SDS-PAGE gel.

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 twice ) and samples were centrifuged for 60 min at 70000 g. The soluble fraction was subjected to further purification by affinity and size exclusion chromatography.
Concentration:20mg/ml
Ligand
MassSpec:
Crystallization:100mM NaAc 4.6, 100mM NaAC, 5% PEG 4K
NMR Spectroscopy:
Data Collection:
Data Processing: