Molecular Biology
Entry Clone Accession: IMAGE:4574938
Entry Clone Source: MGC
SGC Construct ID: GBE1A-c106
Protein Region: L38-L700
Vector: pFB-LIC-Bse
Tag: N-6HIS; TEV cleavage site
Host: DH10Bac
Sequence (with tag(s)): MGHHHHHHSSGVDLGTENLYFQSMLKPYAVDFQRRYKQFSQILKNIGENEGGIDKFSRGYESFGVHRCADGGLYCKEWAPGAEGVFLTGDFNGWNPFSYPYKKLDYGKWELYIPPKQNKSVLVPHGSKLKVVITSKSGEILYRISPWAKYVVREGDNVNYDWIHWDPEHSYEFKHSRPKKPRSLRIYESHVGISSHEGKVASYKHFTCNVLPRIKGLGYNCIQLMAIMEHAYYASFGYQITSFFAASSRYGSPEELQELVDTAHSMGIIVLLDVVHSHASKNSADGLNMFDGTDSCYFHSGPRGTHDLWDSRLFAYSSWEVLRFLLSNIRWWLEEYRFDGFRFDGVTSMLYHHHGVGQGFSGDYSEYFGLQVDEDALTYLMLANHLVHTLCPDSITIAEDVSGMPALCSPISQGGGGFDYRLAMAIPDKWIQLLKEFKDEDWNMGDIVYTLTNRRYLEKCIAYAESHDQALVGDKSLAFWLMDAEMYTNMSVLTPFTPVIDRGIQLHKMIRLITHGLGGEGYLNFMGNEFGHPEWLDFPRKGNNESYHYARRQFHLTDDDLLRYKFLNNFDRDMNRLEERYGWLAAPQAYVSEKHEGNKIIAFERAGLLFIFNFHPSKSYTDYRVGTALPGKFKIVLDSDAAEYGGHQRLDHSTDFFSEAFEHNGRPYSLLVYIPSRVALILQNVDL
Sequence after tag cleavage: SMLKPYAVDFQRRYKQFSQILKNIGENEGGIDKFSRGYESFGVHRCADGGLYCKEWAPGAEGVFLTGDFNGWNPFSYPYKKLDYGKWELYIPPKQNKSVLVPHGSKLKVVITSKSGEILYRISPWAKYVVREGDNVNYDWIHWDPEHSYEFKHSRPKKPRSLRIYESHVGISSHEGKVASYKHFTCNVLPRIKGLGYNCIQLMAIMEHAYYASFGYQITSFFAASSRYGSPEELQELVDTAHSMGIIVLLDVVHSHASKNSADGLNMFDGTDSCYFHSGPRGTHDLWDSRLFAYSSWEVLRFLLSNIRWWLEEYRFDGFRFDGVTSMLYHHHGVGQGFSGDYSEYFGLQVDEDALTYLMLANHLVHTLCPDSITIAEDVSGMPALCSPISQGGGGFDYRLAMAIPDKWIQLLKEFKDEDWNMGDIVYTLTNRRYLEKCIAYAESHDQALVGDKSLAFWLMDAEMYTNMSVLTPFTPVIDRGIQLHKMIRLITHGLGGEGYLNFMGNEFGHPEWLDFPRKGNNESYHYARRQFHLTDDDLLRYKFLNNFDRDMNRLEERYGWLAAPQAYVSEKHEGNKIIAFERAGLLFIFNFHPSKSYTDYRVGTALPGKFKIVLDSDAAEYGGHQRLDHSTDFFSEAFEHNGRPYSLLVYIPSRVALILQNVDL
DNA Sequence: CCATGGGCCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTTGAAGCCCTACGCCGTGGACTTCCAGCGCAGGTATAAGCAGTTTAGCCAAATTTTGAAGAACATTGGAGAAAATGAAGGTGGTATTGATAAGTTTTCCAGAGGCTATGAATCATTTGGCGTCCACAGATGTGCTGATGGTGGTTTATACTGCAAAGAATGGGCCCCGGGAGCAGAAGGAGTTTTTCTTACTGGAGATTTTAATGGTTGGAATCCATTTTCGTACCCATACAAAAAACTGGATTATGGAAAATGGGAGCTGTATATCCCACCAAAGCAGAATAAATCTGTACTCGTGCCTCATGGATCCAAATTAAAGGTAGTTATTACTAGTAAAAGCGGAGAGATCTTGTATCGTATTTCACCGTGGGCAAAGTATGTGGTTCGTGAAGGTGATAATGTGAATTATGATTGGATACACTGGGATCCAGAACACTCATATGAGTTTAAGCATTCCAGACCAAAGAAGCCACGGAGTCTAAGAATTTATGAATCTCATGTGGGAATTTCTTCCCATGAAGGAAAAGTAGCTTCTTATAAACATTTTACATGCAATGTACTACCAAGAATCAAAGGCCTTGGATACAACTGCATTCAGTTGATGGCAATCATGGAGCATGCTTACTATGCCAGCTTTGGTTACCAAATCACAAGCTTCTTTGCAGCTTCCAGCCGTTATGGATCACCTGAAGAGCTACAAGAACTGGTAGACACAGCTCATTCCATGGGTATCATAGTCCTCTTAGATGTGGTACACAGCCATGCTTCAAAAAATTCAGCAGATGGATTGAATATGTTTGATGGGACAGATTCCTGTTATTTTCATTCTGGACCTAGAGGGACTCATGATCTTTGGGATAGCAGATTGTTTGCCTACTCCAGCTGGGAAGTTTTAAGATTCCTTCTGTCAAACATAAGATGGTGGTTGGAAGAATATCGCTTTGATGGATTTCGTTTTGATGGTGTTACGTCCATGCTTTATCATCACCATGGAGTGGGTCAAGGTTTCTCAGGTGATTACAGTGAATATTTCGGACTACAAGTAGATGAAGATGCCTTGACTTACCTCATGTTGGCAAATCATTTGGTTCACACGCTGTGTCCCGATTCTATAACAATAGCTGAGGATGTATCAGGAATGCCAGCTCTGTGCTCTCCAATTTCCCAGGGAGGGGGTGGTTTTGACTATCGACTAGCCATGGCAATTCCAGATAAGTGGATTCAGCTACTTAAAGAGTTTAAAGATGAAGACTGGAACATGGGCGATATAGTATACACGCTCACAAACAGGCGCTACCTTGAAAAGTGCATTGCTTATGCAGAGAGCCATGATCAGGCATTGGTTGGGGATAAGTCGCTGGCATTTTGGTTGATGGATGCCGAAATGTATACAAACATGAGTGTCCTGACTCCTTTTACTCCAGTTATTGATCGTGGAATACAGCTTCATAAAATGATTCGACTCATTACGCATGGGCTTGGTGGAGAAGGCTATCTCAATTTCATGGGTAATGAATTTGGGCATCCTGAATGGTTAGACTTCCCAAGAAAAGGAAATAATGAGAGTTACCATTATGCCAGGCGGCAGTTTCATTTAACTGACGACGACCTTCTTCGCTACAAGTTCCTAAATAATTTTGACAGGGATATGAATAGATTGGAAGAAAGATATGGTTGGCTTGCAGCTCCACAGGCCTACGTGAGTGAAAAACATGAAGGCAATAAGATCATTGCTTTTGAAAGAGCAGGTCTTCTTTTCATTTTCAACTTCCATCCAAGCAAGAGCTACACTGACTACCGAGTTGGAACAGCATTGCCAGGGAAATTCAAAATTGTGCTAGATTCAGATGCAGCGGAATATGGAGGGCATCAGAGACTGGACCACAGCACTGACTTTTTTTCTGAGGCTTTTGAACATAATGGGCGTCCCTATTCTCTTTTGGTGTACATTCCAAGCAGAGTGGCCCTCATCCTTCAGAATGTGGATCTGTGACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTT
Protein Expression
Medium: Sf9 medium
Procedure: High five cells were grown in Sf9 medium at 27°C. Cells were infected at a density of 2x106/ml with recombinant baculovirus (virus stock P2; 1ml of virus stock/1l of cell culture) Culture was supplemented with FCS to final concentration 1%. 120 hours post infection the cultures were collected and centrifuged for 20min at 1500 xg. Cellular pellets were discarded and supernatants were used as a source of a protein.
Protein Purification
Cell Lysis
Cell pellets were dissolved in approximately 50ml lysis buffer and broken by sonication for 10min @ 35% amplitude followed by homogenization by 2 passes at 12,000 psi. The cell debris was pelleted at 40,000 x g and the supernatant used for further purification.
Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 0.5 mM TCEP, 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche)
Column 1
Ni-NTA (1.5 ml volume in a gravity-flow column).
The clarified cell extract was added to 1.5 ml of Ni-NTA pre-equilibrated with lysis buffer and passed through a glass column. The column was then washed with Binding Buffer (2 x 15 ml) and Wash Buffer (2 x 15 ml). The protein was eluted with 25 ml of Elution Buffer in 5 x 2.5 ml fractions.
Binding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 0.5 mM TCEP
Wash Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.4, 0.5 mM TCEP
Elution Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5 mM TCEP
Column 2
Superdex 200 16/60 Gel Filtration.
The first three elution fractions from column 1 were pooled and concentrated to 5 ml with a 30 kDa mwco spin concentrator and injected into an s200 16/60 column (preequilibrated in GF Buffer) at 1.0 ml/min. 1.0 ml fractions were collected.
The protein eluted at between 80 ml and 90 ml volume.
GF Buffer: 10 mM Hepes pH 7.4, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol.
Concentration
Pooled protein fractions were concentrated to 16.5 mg/ml using a 30 kDa mwco concentrator.
Mass-spec Verification: Yes
Measured mass: 78939.2 Da
Expected mass: 79023.3 Da
Structure Determination
Crystallization: Crystals were grown by vapour diffusion in sitting drop at 4°C. A sitting drop consisting of 50 nl protein (pre-incubated with 5 mM acarbose) and 100 nl well solution was equilibrated against well solution containing 0.05 M sodium-succinate and 22% PEG 3350 supplemented with 4% formamide (Hampton additive screen). Crystals were mounted in the presence of 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.
Data collection: Resolution: 2.70 Å; X-ray source: Diamond I04
