Molecular Biology
Entry Clone Accession: NM_017545
Entry Clone Source: Origene
SGC Construct ID: HAO1A-c002
Protein Region: M1-S368
Vector: pNIC28-Bsa4
Tag: N-6HIS; TEV cleavage site
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MHHHHHHSSGVDLGTENLYFQSMLPRLICINDYEQHAKSVLPKSIYDYYRSGANDEETLADNIAAFSRWKLYPRMLRNVAETDLSTSVLGQRVSMPICVGATAMQRMAHVDGELATVRACQSLGTGMMLSSWATSSIEEVAEAGPEALRWLQLYIYKDREVTKKLVRQAEKMGYKAIFVTVDTPYLGNRLDDVRNRFKLPPQLRMKNFETSTLSFSPEENFGDDSGLAAYVAKAIDPSISWEDIKWLRRLTSLPIVAKGILRGDDAREAVKHGLNGILVSNHGARQLDGVPATIDVLPEIVEAVEGKVEVFLDGGVRKGTDVLKALALGAKAVFVGRPIVWGLAFQGEKGVQDVLEILKEEFRLAMALSGCQNVKVIDKTLVRKNPLAVS
Sequence after tag cleavage: SMLPRLICINDYEQHAKSVLPKSIYDYYRSGANDEETLADNIAAFSRWKLYPRMLRNVAETDLSTSVLGQRVSMPICVGATAMQRMAHVDGELATVRACQSLGTGMMLSSWATSSIEEVAEAGPEALRWLQLYIYKDREVTKKLVRQAEKMGYKAIFVTVDTPYLGNRLDDVRNRFKLPPQLRMKNFETSTLSFSPEENFGDDSGLAAYVAKAIDPSISWEDIKWLRRLTSLPIVAKGILRGDDAREAVKHGLNGILVSNHGARQLDGVPATIDVLPEIVEAVEGKVEVFLDGGVRKGTDVLKALALGAKAVFVGRPIVWGLAFQGEKGVQDVLEILKEEFRLAMALSGCQNVKVIDKTLVRKNPLAVS
DNA sequence: CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGCTCCCCCGGCTAATTTGTATCAATGATTATGAACAACATGCTAAATCAGTACTTCCAAAGTCTATATATGACTATTACAGGTCTGGGGCAAATGATGAAGAAACTTTGGCTGATAATATTGCAGCATTTTCCAGATGGAAGCTGTATCCAAGGATGCTCCGGAATGTTGCTGAAACAGATCTGTCGACTTCTGTTTTAGGACAGAGGGTCAGCATGCCAATATGTGTGGGGGCTACGGCCATGCAGCGCATGGCTCATGTGGACGGCGAGCTTGCCACTGTGAGAGCCTGTCAGTCCCTGGGAACGGGCATGATGTTGAGTTCCTGGGCCACCTCCTCAATTGAAGAAGTGGCGGAAGCTGGTCCTGAGGCACTTCGTTGGCTGCAACTGTATATCTACAAGGACCGAGAAGTCACCAAGAAGCTAGTGCGGCAGGCAGAGAAGATGGGCTACAAGGCCATATTTGTGACAGTGGACACACCTTACCTGGGCAACCGTCTGGATGATGTGCGTAACAGATTCAAACTGCCGCCACAACTCAGGATGAAAAATTTTGAAACCAGTACTTTATCATTTTCTCCTGAGGAAAATTTTGGAGACGACAGTGGACTTGCTGCATATGTGGCTAAAGCAATAGACCCATCTATCAGCTGGGAAGATATCAAATGGCTGAGAAGACTGACATCATTGCCAATTGTTGCAAAGGGCATTTTGAGAGGTGATGATGCCAGGGAGGCTGTTAAACATGGCTTGAATGGGATCTTGGTGTCGAATCATGGGGCTCGACAACTCGATGGGGTGCCAGCCACTATTGATGTTCTGCCAGAAATTGTGGAGGCTGTGGAAGGGAAGGTGGAAGTCTTCCTGGACGGGGGTGTGCGGAAAGGCACTGATGTTCTGAAAGCTCTGGCTCTTGGCGCCAAGGCTGTGTTTGTGGGGAGACCAATCGTTTGGGGCTTAGCTTTCCAGGGGGAGAAAGGTGTTCAAGATGTCCTCGAGATACTAAAGGAAGAATTCCGGTTGGCCATGGCTCTGAGTGGGTGCCAGAATGTGAAAGTCATCGACAAGACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCTGACAGTAAAGGTGGATACGGATCCGAA
Protein Expression
Medium: Terrific Broth
Antibiotics: Kanamycin
Procedure: An overnight culture (20 mL LB) was used to inoculate 2L auto-induction TB containing 50 µg/ml each of kanamycin and chloramphenicol. Cells were cultured at 37°C for 6 hours followed by 48 h incubation at 18°C.
Protein Purification
Procedure: Cell pellet from 2 L of culture was re-suspended in 150 mL Extraction Buffer, lysed by sonication for 15 minutes, and centrifuged at 37000 x g for 1 hour at 4°C. The clarified cell extract was incubated with 5 mL of Ni-NTA resin pre-equilibrated with lysis buffer before applying to 1.5 x 10 cm column by gravity flow. The column was washed with 10 column volumes of Binding Buffer, 10 column volumes of Wash Buffer, and eluted with 5 x 5 mL Elution Buffer. Fractions containing hHAO1 were loaded onto gel filtration column (Superdex 200 Hiload 16/60) pre-equilibrated with GF buffer. Fractions containing hHAO1 were pooled.
Extraction Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol, 1:1000 of Merck Protease Cocktail II, 0.5 mg/mL lysozyme and 0.2 µg/mL benzonase.
Binding Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol.
Washing Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 40 mM imidazole, 0.5 mM TCEP, 5% glycerol.
Elution Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 250 mM imidazole, 0.5 mM TCEP, 5% glycerol.
GF buffer: 500 mM NaCl, 550 mM HEPES pH 7.5, 0.5 mM TCEP, 5% glycerol.
Concentration: 13.7 mg/ml
Mass-spec Verification: Yes
Structure Determination
Crystallization: Crystals were grown from hHAO1 protein pre-incubated with 2 mM CCPST, equilibrated against well solution containing 30% PEG1000, sodium malonate-imidazole-boric acid pH 8.0. Crystal was cryo-protected with 20-25% ethylene glycol before flash-cooling in liquid nitrogen.
Data Collection: Beamline: Dmnd I03; Resolution: 1.2 Å
Data Processing: hHAO1 structure was solved by molecular replacement with PHASER using 2NZL as the search model, followed by iterative cycles of REFMAC5 including TLS refinement, and manual model building in Coot.
