Molecular Biology
Entry Clone Accession: IMAGE 3493526
Entry Clone Source: IMAGE
SGC Construct ID: MMADHCMMA-c001
Protein Region: P129-N296
Vector: pNIC28-Bsa4
Tag: N-6HIS;N-TEV
Host: BL21(DE3)-R3
Sequence (with tag(s)): MHHHHHHSSGVDLGTENLYFQSMPVEQEINSAETYFESAKVECAIQTCPELLRRDFESLFPEVANSKLMILTVTQKTENDMTVWSEEVEVEREVLLEKFISGAKEICYALRAEGYWADFIDPSSGVAFFGPYTNNTLFETDERYRHLGFSVDDLGCCKVIRHSLWGTHVVVGSIFTNATADSSIMRKLSGN
Sequence after tag cleavage: SMPVEQEINSAETYFESAKVECAIQTCPELLRRDFESLFPEVANSKLMILTVTQKTENDMTVWSEEVEVEREVLLEKFISGAKEICYALRAEGYWADFIDPSSGVAFFGPYTNNTLFETDERYRHLGFSVDDLGCCKVIRHSLWGTHVVVGSIFTNATADSSIMRKLSGN
DNA Sequence: CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGCCTGTTGAACAAGAAATTAACAGTGCAGAAACTTACTTTGAAAGTGCCAAAGTTGAGTGTGCGATCCAGACATGTCCAGAGCTGCTGCGGAGAGATTTTGAGTCACTTTTTCCAGAAGTGGCTAATAGCAAACTAATGATCCTGACTGTAACCCAGAAAACTGAAAATGACATGACTGTCTGGAGTGAAGAGGTGGAGGTTGAGAGAGAGGTGCTCTTGGAAAAGTTCATCAGTGGTGCTAAGGAGATCTGCTATGCCCTTCGGGCTGAAGGATATTGGGCTGACTTCATTGACCCATCATCTGGTGTGGCATTTTTTGGACCATATACCAACAACACACTTTTTGAAACAGATGAACGGTACCGGCATTTAGGATTCTCAGTTGACGACCTTGGCTGCTGTAAAGTGATCCGTCACAGTCTCTGGGGTACTCATGTGGTTGTAGGAAGTATCTTCACTAACGCAACAGCAGACAGCAGTATCATGAGGAAGCTCAGTGGAAACTAGCAGTAAAGGTGGATACGGATCCGAA
Protein Expression
Medium: TB
Antibiotics: Kanamycin
Procedure: Protein was expressed in BL21(DE3)R3-Rosetta E. coli cells grown in TB medium with 50 μg/ml kanamycin, with induction of IPTG overnight at 18°C. Cell pellets were harvested, sonicated in lysis buffer (50 mM HEPES pH7.5, 10 mM imidazole, 500 mM NaCl, 5% glycerol) and insoluble material removed by centrifugation.
Protein Purification
Procedure: The supernatant was purified by affinity (Ni-Sepharose; GE Healthcare) and size-exclusion (Superdex 200; GE Healthcare) chromatography.
Concentration: 23.0 mg/ml
Mass-spec Verification: Yes
Structure Determination
Crystallization: SeMet crystals of mouse MMADHC were grown by vapor diffusion at 4°C in sitting drops containing 100 nl of protein (23 mg/ml) and 50 nl mother liquor (28% PEG3350, 100 mM bis-tris pH 6.5, 250 mM NaCl). Native crystals were grown by vapor diffusion at 4°C in sitting drops containing 75 nl of protein (23 mg/ml) and 75 nl mother liquor (22% polyacrylic acid 5100, 100 mM HEPES pH 7.5, 20 mM MgCl2). Crystals were cryo-protected in mother liquor containing ethylene glycol (25% v/v) and flash-cooled in liquid nitrogen.
Protein Concentration: 23.0mg/ml
Data Collection: Beamline: Dmnd I04; Resolution: 2.3 Å; Diffraction data were collected at the Diamond Light Source beamlines I04, and processed using xia2.
