Molecular Biology
Entry Clone Accession: MGC:9490 IMAGE:3922902; note this clone harbours the p.Asn314Asp polymorphism
Entry Clone Source: MGC
SGC Construct ID: GALTA-c001
Protein Region: M1-A379
Vector: pNIC28-Bsa4
Tag: N-6HIS;N-TEV
Host: BL21(DE3)-R3-pRARE2.
Sequence (with tag(s)): MHHHHHHSSGVDLGTENLYFQSMSRSGTDPQQRQQASEADAAAATFRANDHQHIRYNPLQDEWVLVSAHRMKRPWQGQVEPQLLKTVPRHDPLNPLCPGAIRANGEVNPQYDSTFLFDNDFPALQPDAPSPGPSDHPLFQAKSARGVCKVMCFHPWSDVTLPLMSVPEIRAVVDAWASVTEELGAQYPWVQIFENKGAMMGCSNPHPHCQVWASSFLPDIAQREERSQQAYKSQHGEPLLMEYSRQELLRKERLVLTSEHWLVLVPFWATWPYQTLLLPRRHVRRLPELTPAERDDLASIMKKLLTKYDNLFETSFPYSMGWHGAPTGSEAGANWDHWQLHAHYYPPLLRSATVRKFMVGYEMLAQAQRDLTPEQAAERLRALPEVHYHLGQKDRETATIA
Sequence after tag cleavage: SMSRSGTDPQQRQQASEADAAAATFRANDHQHIRYNPLQDEWVLVSAHRMKRPWQGQVEPQLLKTVPRHDPLNPLCPGAIRANGEVNPQYDSTFLFDNDFPALQPDAPSPGPSDHPLFQAKSARGVCKVMCFHPWSDVTLPLMSVPEIRAVVDAWASVTEELGAQYPWVQIFENKGAMMGCSNPHPHCQVWASSFLPDIAQREERSQQAYKSQHGEPLLMEYSRQELLRKERLVLTSEHWLVLVPFWATWPYQTLLLPRRHVRRLPELTPAERDDLASIMKKLLTKYDNLFETSFPYSMGWHGAPTGSEAGANWDHWQLHAHYYPPLLRSATVRKFMVGYEMLAQAQRDLTPEQAAERLRALPEVHYHLGQKDRETATIA
DNA Sequence: CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTCGCGCAGTGGAACCGATCCTCAGCAACGCCAGCAGGCGTCAGAGGCGGACGCCGCAGCAGCAACCTTCCGGGCAAACGACCATCAGCATATCCGCTACAACCCGCTGCAGGATGAGTGGGTGCTGGTGTCAGCTCACCGCATGAAGCGGCCCTGGCAGGGTCAAGTGGAGCCCCAGCTTCTGAAGACAGTGCCCCGCCATGACCCTCTCAACCCTCTGTGTCCTGGGGCCATCCGAGCCAACGGAGAGGTGAATCCCCAGTACGATAGCACCTTCCTGTTTGACAACGACTTCCCAGCTCTGCAGCCTGATGCCCCCAGTCCAGGACCCAGTGATCATCCCCTTTTCCAAGCAAAGTCTGCTCGAGGAGTCTGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTGATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGATCTTTGAAAACAAAGGTGCCATGATGGGCTGTTCTAACCCCCACCCCCACTGCCAGGTATGGGCCAGCAGTTTCCTGCCAGATATTGCCCAGCGTGAGGAGCGATCTCAGCAGGCCTATAAGAGTCAGCATGGAGAGCCCCTGCTAATGGAGTACAGCCGCCAGGAGCTACTCAGGAAGGAACGTCTGGTCCTAACCAGTGAGCACTGGTTAGTACTGGTCCCCTTCTGGGCAACATGGCCCTACCAGACACTGCTGCTGCCCCGTCGGCATGTGCGGCGGCTACCTGAGCTGACCCCTGCTGAGCGTGATGATCTAGCCTCCATCATGAAGAAGCTCTTGACCAAGTATGACAACCTCTTTGAGACGTCCTTTCCCTACTCCATGGGCTGGCATGGGGCTCCCACAGGATCAGAGGCTGGGGCCAACTGGGACCATTGGCAGCTGCACGCTCATTACTACCCTCCGCTCCTGCGCTCTGCCACTGTCCGGAAATTCATGGTTGGCTACGAAATGCTTGCTCAGGCTCAGAGGGACCTCACCCCTGAGCAGGCTGCAGAGAGACTAAGGGCACTTCCTGAGGTTCATTACCACCTGGGGCAGAAGGACAGGGAGACAGCAACCATCGCCTGACAGTAAAGGTGGATACGGATCCGAA
Protein Expression
hGALT (wt or variants) was cultured in 6 L of Terrific Broth at 37 °C, and induced with 0.1 mM IPTG overnight at 18 °C. Cell pellets were harvested, homogenized in lysis buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) and centrifuged to remove insoluble material.
Protein Purification
The supernatant was purified by immobilized metal affinity (Talon resin; GE Healthcare) and size-exclusion chromatography in Superdex 200 Hi-Load 16/60 column (GE Healthcare), pre-equilibrated with buffer 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. Purified protein was treated with His-tagged TEV protease overnight at 4 °C, and further purified by reverse Nickel affinity and size exclusion chromatography. The final purified protein was concentrated to 20 mg∕ml, flash cooled in liquid nitrogen and stored at −80 °C.
Concentration: 20 mg/ml
Mass-spec Verification: yes
Structure Determination
Crystallization: Crystals were grown by vapour diffusion at 20 °C, from sitting drops mixing 200 nl of protein (20 mg/ml; pre-incubated with 5 m M UDP-Glc) and 100 nl of reservoir solution containing 0.2 M ammonium sulphate, 30% (w/v) PEG 8000. Crystals were cryo-protected with reservoir solution supplemented with 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. The structures were solved by molecular replacement with PHASER, using the structure of E. coli GALT (1HXP) as template. Modelling and refinement were carried out using Refmac and Coot.
Data Collection: Beamline: Dmnd I02; Resolution: 1.73 Å
