Molecular Biology
Entry Clone Accession: IMAGE:5744956
Entry Clone Source: MGC
SGC Construct ID: HMGCS2A-c010
Protein Region: P51-V508
Vector: pNH-TrxT
Tag: N-6HIS;N-TRX;N-TEV
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MHHHHHHSSGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGTENLYFQSMPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTRMGFCSVQEDINSLCLTVVQRLMERIQLPWDSVGRLEVGTETIIDKSKAVKTVLMELFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGRYAMVVCGDIAVYPSGNARPTGGAGAVAMLIGPKAPLALERGLRGTHMENVYDFYKPNLASEYPIVDGKLSIQCYLRALDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQYMIFHTPFCKMVQKSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKDLDKALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLASLLSHHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSSTSDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLFPGTWYLERVDEQHRRKYARRPV
Sequence after tag cleavage: SMPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTRMGFCSVQEDINSLCLTVVQRLMERIQLPWDSVGRLEVGTETIIDKSKAVKTVLMELFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGRYAMVVCGDIAVYPSGNARPTGGAGAVAMLIGPKAPLALERGLRGTHMENVYDFYKPNLASEYPIVDGKLSIQCYLRALDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQYMIFHTPFCKMVQKSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKDLDKALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLASLLSHHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSSTSDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLFPGTWYLERVDEQHRRKYARRPV
DNA Sequence: CATATGCACCATCATCATCATCATTCTTCTGGTATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGCGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTACCGAGAACTTGTACTTCCAATCCATGCCAAAGGACGTGGGCATCCTGGCCCTGGAGGTCTACTTCCCAGCCCAATATGTGGACCAAACTGACCTGGAGAAGTATAACAATGTGGAAGCAGGAAAGTATACAGTGGGCTTGGGCCAGACCCGTATGGGCTTCTGCTCAGTCCAAGAGGACATCAACTCCCTGTGCCTGACGGTGGTGCAACGGCTGATGGAGCGCATACAGCTCCCATGGGACTCTGTGGGCAGGCTGGAAGTAGGCACTGAGACCATCATTGACAAGTCCAAAGCTGTCAAAACAGTGCTCATGGAACTCTTCCAGGATTCAGGCAATACTGATATTGAGGGCATAGATACCACCAATGCCTGCTACGGTGGTACTGCCTCCCTCTTCAATGCTGCCAACTGGATGGAGTCCAGTTCCTGGGATGGTCGTTATGCCATGGTGGTCTGTGGAGACATTGCCGTCTATCCCAGTGGTAATGCTCGTCCCACAGGTGGGGCCGGAGCTGTGGCTATGCTGATTGGGCCCAAGGCCCCTCTGGCCCTGGAGCGAGGGCTGAGGGGAACCCATATGGAGAATGTGTATGACTTCTACAAACCAAATTTGGCCTCGGAGTACCCAATAGTGGATGGGAAGCTTTCCATCCAGTGCTACTTGCGGGCCTTGGATCGATGTTACACATCATACCGTAAAAAAATCCAGAATCAGTGGAAGCAAGCTGGCAGCGATCGACCCTTCACCCTTGACGATTTACAGTACATGATCTTTCATACACCCTTTTGCAAGATGGTCCAGAAGTCTCTGGCTCGCCTGATGTTCAATGACTTCCTGTCAGCCAGCAGTGACACACAAACCAGCTTATATAAGGGGCTGGAGGCTTTCGGGGGGCTAAAGCTGGAAGACACCTACACCAACAAGGACCTGGATAAAGCACTTCTAAAGGCCTCTCAGGACATGTTCGACAAGAAAACCAAGGCTTCCCTTTACCTCTCCACTCACAATGGGAACATGTACACCTCATCCCTGTACGGGTGCCTGGCCTCGCTTCTGTCCCACCACTCTGCCCAAGAACTGGCTGGCTCCAGGATTGGTGCCTTCTCTTATGGCTCTGGTTTAGCAGCAAGTTTCTTTTCATTTCGAGTATCCCAGGATGCTGCTCCAGGCTCTCCCCTGGACAAGTTGGTGTCCAGCACATCAGACCTGCCAAAACGCCTAGCCTCCCGAAAGTGTGTGTCTCCTGAGGAGTTCACAGAAATAATGAACCAAAGAGAGCAATTCTACCATAAGGTGAATTTCTCCCCACCTGGTGACACAAACAGCCTTTTCCCAGGTACTTGGTACCTGGAGCGAGTGGACGAGCAGCATCGCCGAAAGTATGCCCGGCGTCCCGTCTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTT
Protein Expression
Medium: TB
Antibiotics: Kanamycin
Procedure: The plasmid was transformed into E. coli BL21(DE3)-R3, cultured in 1 L Terrific Broth at 37°C to OD600 ~1.5, and induced with 0.5 mM IPTG overnight at 18oC. Harvested cells were homogenized in buffer A (50 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole) and centrifuged.
Protein Purification
Procedure: The supernatant was applied to Ni-sepharose resin pre-equilibrated with buffer A, and eluted with buffer B (buffer A + 230 mM imidazole). The eluant was applied to Superdex S200 column pre-equilibrated with buffer GF (10 mM Hepes pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP). To remove fusion tag, purified protein was treated with His-tagged TEV protease overnight at 4°C, and passed over Ni-sepharose resin equilibrated with buffer GF. Protein was concentrated to 12 mg/ml (hHMGCS2).
Concentration: 12.0 mg/ml
Mass-spec Verification: yes
Structure Determination
Crystallization: Protein was pre-incubated with 5 mM Ac-CoA and 5 mM AcAc-CoA prior to crystallization. Sitting drops containing 100 nL protein and 50 nL reservoir solution (0.2 M ammonium sulphate, 0.1 M Bis-Tris pH 6.5, 25% (w/v) PEG 3350) were equilibrated at 20°C. Crystals were cryo-protected using 25% glycerol and flash-cooled in liquid nitrogen. Diffraction data were collected at the SLS beamline X10S, and processed using the CCP4 program suite.
Data Collection: Beamline: SLS-X10; Resolution: 1.75 Å
The structure was solved by molecular replacement using the refined hHMGCS1 structure as search model. Automated model building was performed with ARP/wARP, followed by iterative cycles of restrained refinement with medium NCS restraints and model building using COOT and PHENIX.
