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Structure
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GID4 fragment in complex with a peptide
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PDB Code
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6CDG, 6CDC, 6CD9, 6CD8, 6CCU, 6CCT
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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-A04
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Tag
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N-terminal tag: MHHHHHHSSGRENLYFQG
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Construct sequence
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MHHHHHHSSGRENLYFQG
SGSKFRGHQKSKGNSYDVEVVLQHVDTGNSYLCGYLKIKGLTEEYPTLTTFFEGEIISKKHPFLTRKWDADEDVDRKHWGK
FLAFYQYAKSFNSDDFDYEELKNGDYVFMRWKEQFLVPDHTIKDISGASFAGFYYICFQKSAASIEGYYYHRSSEWYQSLNLTHV
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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.
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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 purified GID4 (aa 124-289) proteins were separately incubated with different peptides at a molar ratio of 1 : 1.5 for 1 h on ice before setting up the crystallization trials. The crystals of GID4-PGLW, GID4-PSRW and GID4-PTLV were grown in the precipitant conditions containing 20-25% (v/v) PEG3350, 2-3% (v/v) Tacsimate, pH 7.0 and 0.1 M HEPES, pH 7.5 (Average pH 7.4). The GID4-PGLWKS was crystallized in 20% (v/v) PEG3350, 0.2 M NaBr. The GID4-PSRV was crystallized in 30% (v/v) PEG2000 and 0.1 M KSCN. The GID4-PHRV was crystallized in 20% (v/v) PEG3350 and 0.03 M Citric acid. The crystals were protected in cryoprotectant solution consisting of reservoir solution supplemented with 20% (v/v) glycerol or 20% (v/v) ethylene glycol and flash-frozen in liquid nitrogen before data collection.
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