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Structure
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Selenomethionyl derivative of a GID4 fragment
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PDB Code
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6CCR
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Entry clone accession
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BC041829.1
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Entry clone source
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MGC:43491 IMAGE:5268071
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SGC clone accession
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JMC130-A06
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Tag
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N-terminal tag: MHHHHHHSSGRENLYFQG
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Construct sequence
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MHHHHHHSSGRENLYFQG
GVATSLLYSGSKFRGHQKSKGNSYDVEVVLQHVDTGNSYL
CGYLKIKGLTEEYPTLTTFFEGEIISKKHPFLTRKWDADEDVD
RKHWGKFLAFYQYAKSFNSDDFDYEELKNGDYVFMRWKE
QFLVPDHTIKDISGASFAGFYYICFQKSAASIEGYYYHRSSEWYQSLNLTHVPEHSAPIYEFR
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Vector
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pET28-MHL
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Expression host
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BL21 (DE3) Codon plus RIL (Stratagene)
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Growth method
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GID4 was 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. To prepare selenomethionine (SeMet) derivatives, SeMet-GID4 (aa 116-300) was overexpressed in the prepacked M9 SeMet growth media kit (Medicilon) following manufacturer’s instructions.
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Extraction buffers
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Lysis buffer: 20 mM Tris-HCl pH 7.5, 400 mM NaCl, 5% glycerol and 2 mM beta-mercaptoethanol
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Extraction procedure
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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.
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Purification buffers
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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 0.5 mM TCEP
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Purification procedure
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The fusion proteins were purified by Ni-NTA agarose column. The His tag was cleaved by His-tagged TEV protease (purified in-house) with an approximate molar ratio of 1 : 20 in the dialysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 5 mM beta-mercaptoethanol) at 4 °C overnight. The tag and protease were removed by reloading onto the Ni-NTA. The proteins were further purified by Superdex 200 10/300 (GE Healthcare). The gel filtration buffer contains 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 0.5 mM TCEP
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Protein stock concentration
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The purified protein was concentrated to 8 mg mL-1 using 15 mL concentrators with a 3,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore).
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Crystallization
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The crystals were grown at 18 °C using sitting drop method by mixing 1 microl protein and 1 microl reservoir solution (10% (v/v) 2-propanol, 20% (v/v) PEG4000 and 0.1 M Na-HEPES, pH 7.5). The SeMet-labelled proteins were obtained under similar crystallization conditions of wild type.
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