Molecular Biology
Entry Clone Accession: NM_199420.3
Entry Clone Source: Remko Prevo (ROB, ORCRB), originally from Richard Wood, Uni of Texas
SGC Construct ID: POLQA-c232
Protein Region: D200-M1029
Vector: pFB-LIC-Bse. This is a baculovirus transfer vector (Bac-to-bac), with N-terminal 6 His tag followed by a TEV cleavage site
Host: DH10Bac
Sequence (with tag(s)): MGHHHHHHSSGVDLGTENLYFQSMDKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLLLGQVLEGKNLVYSAPTSAGKTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSLFQEVGIKVDGYMGSTSPSRHFSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELHMLGDSHRGYLLELLLTKICYITRKSASCQADLASSLSNAVQIVGMSATLPNLELVASWLNAELYHTDFRPVPLLESVKVGNSIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETICDNHSVLLFCPSKKWCEKLADIIAREFYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRLPSGLDSVLQKTVPWGVAFHHAGLTFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRVIIRTPIFGGRPLDILTYKQMVGRAGRKGVDTVGESILICKNSEKSKGIALLQGSLKPVRSCLQRREGEEVTGSMIRAILEIIVGGVASTSQDMHTYAACTFLAASMKEGKQGIQRNQESVQLGAIEACVMWLLENEFIQSTEASDGTEGKVYHPTHLGSATLSSSLSPADTLDIFADLQRAMKGFVLENDLHILYLVTPMFEDWTTIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLARCVKGKVVARTERQHRQMAIHKRFFTSLVLLDLISEVPLREINQKYGCNRGQIQSLQQSAAVYAGMITVFSNRLGWHNMELLLSQFQKRLTFGIQRELCDLVRVSLLNAQRARVLYASGFHTVADLARANIVEVEVILKNAVPFKSARKAVDEEEEAVEERRNMRTIWVTGRKGLTEREAAALIVEEARMILQQDLVEM
Sequence after tag cleavage: SMDKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLLLGQVLEGKNLVYSAPTSAGKTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSLFQEVGIKVDGYMGSTSPSRHFSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELHMLGDSHRGYLLELLLTKICYITRKSASCQADLASSLSNAVQIVGMSATLPNLELVASWLNAELYHTDFRPVPLLESVKVGNSIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETICDNHSVLLFCPSKKWCEKLADIIAREFYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRLPSGLDSVLQKTVPWGVAFHHAGLTFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRVIIRTPIFGGRPLDILTYKQMVGRAGRKGVDTVGESILICKNSEKSKGIALLQGSLKPVRSCLQRREGEEVTGSMIRAILEIIVGGVASTSQDMHTYAACTFLAASMKEGKQGIQRNQESVQLGAIEACVMWLLENEFIQSTEASDGTEGKVYHPTHLGSATLSSSLSPADTLDIFADLQRAMKGFVLENDLHILYLVTPMFEDWTTIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLARCVKGKVVARTERQHRQMAIHKRFFTSLVLLDLISEVPLREINQKYGCNRGQIQSLQQSAAVYAGMITVFSNRLGWHNMELLLSQFQKRLTFGIQRELCDLVRVSLLNAQRARVLYASGFHTVADLARANIVEVEVILKNAVPFKSARKAVDEEEEAVEERRNMRTIWVTGRKGLTEREAAALIVEEARMILQQDLVEM
DNA Sequence: ATGGGCCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGACAAGCTACTATTGGCAAACTGGGGACTTCCTAAAGCAGTTCTGGAAAAATACCACAGTTTTGGTGTAAAAAAGATGTTTGAATGGCAGGCAGAGTGCCTTTTGCTTGGACAAGTCCTGGAAGGAAAGAATTTAGTTTATTCAGCTCCTACAAGTGCTGGGAAGACTCTTGTGGCAGAATTACTTATTTTGAAGCGGGTTTTGGAAATGCGGAAGAAAGCTTTGTTTATTCTTCCCTTTGTTTCTGTGGCTAAAGAGAAGAAATACTACCTCCAGAGTCTGTTTCAGGAAGTAGGAATAAAAGTAGACGGTTATATGGGCAGCACCTCTCCATCAAGGCATTTCTCTTCATTGGATATTGCAGTCTGCACAATTGAGAGAGCCAATGGTCTGATCAATCGCCTCATAGAGGAAAATAAGATGGATCTGTTAGGAATGGTGGTTGTGGATGAATTACATATGCTGGGAGACTCTCACCGAGGGTATCTGCTGGAACTTTTGCTGACCAAGATTTGCTATATTACTCGGAAATCAGCATCTTGTCAGGCAGATCTAGCCAGTTCTCTGTCTAATGCTGTGCAAATCGTTGGCATGAGTGCTACCCTTCCTAATTTGGAGCTTGTGGCTTCCTGGTTGAATGCTGAACTCTACCATACCGACTTTCGCCCTGTACCGCTTTTGGAGTCAGTAAAAGTTGGAAATTCCATATATGACTCTTCAATGAAACTTGTGAGGGAATTTGAGCCCATGCTTCAAGTGAAGGGAGATGAGGACCATGTTGTTAGCTTATGTTATGAGACGATTTGTGATAACCATTCAGTATTACTTTTTTGTCCATCAAAGAAATGGTGTGAGAAGCTGGCAGATATCATTGCTCGAGAGTTTTATAATCTACATCATCAAGCTGAGGGATTGGTGAAACCCTCTGAATGCCCACCAGTAATTCTGGAACAAAAAGAACTCCTGGAAGTGATGGATCAGTTAAGACGGTTGCCTTCAGGACTGGACTCTGTATTACAGAAAACTGTACCATGGGGAGTAGCATTTCATCATGCAGGTCTTACTTTTGAGGAGAGGGATATCATTGAAGGAGCCTTTCGTCAAGGTCTCATTCGAGTCTTGGCGGCAACTTCTACTCTTTCTTCTGGGGTGAATTTACCTGCACGTCGTGTGATTATTCGAACCCCTATTTTTGGTGGTCGACCTCTAGATATTCTTACTTATAAGCAGATGGTTGGTCGTGCTGGCAGGAAAGGAGTGGACACAGTAGGCGAGAGTATCTTAATTTGTAAGAACTCTGAGAAATCAAAAGGCATAGCTCTCCTTCAGGGTTCTCTAAAGCCTGTTCGCAGCTGTCTGCAAAGACGAGAAGGAGAAGAAGTAACTGGCAGCATGATACGAGCTATTCTGGAGATAATAGTTGGTGGAGTGGCAAGTACATCACAAGATATGCATACTTATGCTGCCTGCACATTTTTGGCTGCAAGTATGAAAGAAGGGAAGCAAGGAATTCAGAGAAATCAAGAGTCTGTTCAGCTTGGAGCGATTGAGGCCTGTGTGATGTGGCTACTAGAAAATGAATTCATCCAGAGTACAGAAGCCAGTGATGGAACAGAAGGAAAGGTGTATCATCCAACACATCTTGGTTCGGCCACTCTTTCTTCTTCACTTTCTCCAGCTGATACTTTAGATATTTTTGCTGACCTGCAAAGAGCAATGAAGGGCTTTGTTTTAGAGAATGATCTTCATATTCTCTATCTGGTTACACCTATGTTTGAGGATTGGACTACTATTGATTGGTATCGATTTTTCTGTTTATGGGAGAAGTTGCCAACTTCAATGAAAAGGGTGGCAGAGCTAGTGGGAGTTGAAGAGGGGTTCTTGGCCCGTTGTGTGAAAGGAAAAGTAGTAGCCAGAACTGAGAGACAGCATCGACAAATGGCCATCCATAAAAGGTTTTTCACCAGTCTTGTGCTATTAGATTTAATCAGTGAAGTTCCCTTAAGGGAAATAAATCAGAAATATGGATGCAATCGTGGGCAGATTCAATCTTTGCAACAGTCAGCTGCTGTTTATGCAGGGATGATTACAGTATTTTCCAACCGTCTGGGCTGGCACAACATGGAACTACTACTTTCCCAATTTCAGAAGCGTCTTACGTTTGGCATCCAGAGGGAGCTGTGTGACCTGGTTCGGGTATCCTTACTAAATGCTCAGAGAGCCAGGGTTCTCTATGCTTCTGGCTTTCATACTGTGGCAGACCTTGCTAGAGCAAATATTGTGGAGGTGGAGGTGATTCTGAAAAATGCTGTGCCTTTCAAAAGTGCCCGGAAGGCAGTGGATGAGGAAGAGGAAGCAGTTGAAGAACGTCGCAATATGCGAACTATCTGGGTGACTGGCAGAAAAGGTTTAACTGAAAGGGAAGCAGCAGCCCTTATAGTGGAAGAAGCCAGAATGATTCTGCAGCAGGACTTAGTTGAAATGTGA
Protein Expression
Medium: SF900II
Antibiotics: Ampicillin
Procedure: Baculoviruses were generated by recombination in E. coli DH10Bac (Life Technologies) followed by transfection and two rounds of amplification in SF9 cells. POLQ was expressed in 1-L cultures of SF9 cells in 4-L shaker flasks at 27°C, infected at 2 × 106 cells/ml with 3 ml of P2 virus, and incubated for further 70 h. The cells were collected by centrifugation, suspended in 15 ml/l of lysis buffer (50 mM HEPES, pH 7.5, 0.5 M NaCl, 5% v/v glycerol, 10 mM imidazole, and 1 mM TCEP) and frozen at −80°C.
Protein Purification
Procedure: Cell pellets were thawed and resuspended in buffer A (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphene (TCEP)), with the addition of 1x protease inhibitor set VII (Merck, Darmstadt, Germany). Cells were lysed using sonication and cell debris pelleted by centrifugation. Lysates were applied to a Ni-IDA IMAC gravity flow column, washed with 2 column volumes of wash buffer (buffer A supplemented with 30 mM imidazole), and eluted with the addition of 300 mM imidazole in buffer A. The purification tag was cleaved with the addition of 1:20 mass ratio of His-tagged TEV protease during overnight dialysis into buffer B (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP). TEV was removed by IMAC column rebinding and final protein purification was performed by size exclusion chromatography using a HiLoad 16/60 Superdex 200 column at 1 ml/min in buffer B. Protein concentrations were determined by measurement at 280nm (Nanodrop) using the calculated molecular mass and extinction coefficients.
The protein was analyzed by ESI-TOF intact mass spectrometry which revealed a major peak at 93102.9 Da which corresponds to the expected mass (93085.5 Da) with oxidation +16 Da.
Structure Determination
Crystallization: For crystallization POLQ was concentrated to 10 mg/ml using a 50,000 mwco centrifugal concentrator and exchanged to a buffer containing 10 mM HEPES pH 7.5, 250 mM NaCl, 0.5 mM TCEP. ADP and MgCl were added to 10 mM final concentration and crystals were obtained at 20°C from conditions containing 20% PEG 3350, 0.1M potassium citrate tribasic pH 8.5. Crystals were cryo-protected by transferring to a solution of mother liquor supplemented with 25 % ethelene glycol and flash-cooled in liquid nitrogen.
Data Collection: Data were collected at a wavelength of 0.92Å at Diamond light source beamline I04- 1 to a resolution of 3.2Å. Diffraction data were processed with the program XDS.
Data Processing: The structures were solved by molecular replacement using the program PHASER with PDBid 2ZJA as a starting model. Model building and real space refinement were performed in COOT and the structures refined using PHENIX REFINE to a final Rfactor= 22.6%, Rfree= 27.3%.
