Molecular Biology
Entry Clone Accession:
Entry Clone Source: Tony Pawson
SGC Construct ID: WNK3B-c005
Protein Region: E132-E414
Vector: pFB-LIC-Bse
Tag: N-6HIS;N-TEV
Host: DH10Bac
Sequence (with tag(s)): MGHHHHHHSSGVDLGTENLYFQSMEAEMKAVATSPSGRFLKFDIELGRGAFKTVYKGLDTETWVEVAWCELQDRKLTKAEQQRFKEEAEMLKGLQHPNIVRFYDSWESILKGKKCIVLVTELMTSGTLKTYLKRFKVMKPKVLRSWCRQILKGLQFLHTRTPPIIHRDLKCDNIFITGPTGSVKIGDLGLATLMRTSFAKSVIGTPEFMAPEMYEEHYDESVDVYAFGMCMLEMATSEYPYSECQNAAQIYRKVTSGIKPASFNKVTDPEVKEIIEGCIRQNKSERLSIRDLLNHAFFAEDTGLRVE
Sequence after tag cleavage: SMEAEMKAVATSPSGRFLKFDIELGRGAFKTVYKGLDTETWVEVAWCELQDRKLTKAEQQRFKEEAEMLKGLQHPNIVRFYDSWESILKGKKCIVLVTELMTSGTLKTYLKRFKVMKPKVLRSWCRQILKGLQFLHTRTPPIIHRDLKCDNIFITGPTGSVKIGDLGLATLMRTSFAKSVIGTPEFMAPEMYEEHYDESVDVYAFGMCMLEMATSEYPYSECQNAAQIYRKVTSGIKPASFNKVTDPEVKEIIEGCIRQNKSERLSIRDLLNHAFFAEDTGLRVE
DNA Sequence: ATGGCCACTGATTCAGGGGATCCAGCCAGCACAGAAGATTCTGAGAAACCTGATGGAATTTCATTTGAAAACAGAGTTCCCCAGGTCGCTGCAACTTTGACAGTAGAAGCTAGACTAAAGGAGAAAAACAGTACCTTCTCTGCTTCTGGGGAAACTGTAGAAAGGAAGAGATTTTTCCGAAAGAGTGTTGAAATGACGGAAGATGACAAAGTTGCCGAATCATCCCCCAAAGATGAGAGAATTAAGGCTGCAATGAATATTCCAAGAGTAGATAAGCTTCCTTCAAATGTGTTGAGAGGTGGACAAGAAGTTAAATATGAACAGTGTTCAAAGTCAACCTCAGAAATCTCAAAAGATTGTTTCAAGGAGAAAAATGAAAAGGAAATGGAAGAAGAAGCAGAAATGAAGGCTGTAGCTACTTCTCCTAGTGGCAGATTCCTGAAATTTGACATAGAACTAGGAAGAGGAGCATTTAAAACAGTATATAAAGGACTGGACACTGAAACATGGGTTGAGGTTGCTTGGTGTGAGCTGCAGGACCGAAAGTTAACCAAAGCTGAGCAGCAAAGATTCAAGGAAGAAGCAGAGATGTTGAAGGGTCTCCAGCACCCCAATATAGTTCGATTTTATGATTCCTGGGAATCTATATTAAAAGGAAAGAAATGTATTGTATTAGTGACTGAACTAATGACATCTGGGACCTTAAAGACGTACTTAAAACGATTTAAAGTCATGAAACCAAAGGTCTTAAGGAGCTGGTGCAGGCAAATTTTAAAGGGGTTGCAGTTCTTGCACACTAGGACTCCTCCTATTATTCACCGGGATCTGAAGTGTGACAATATTTTCATCACGGGACCCACTGGATCTGTGAAGATTGGTGATCTAGGATTAGCCACCTTAATGCGTACCTCATTTGCTAAGAGTGTCATTGGAACTCCTGAGTTTATGGCTCCAGAGATGTATGAAGAACACTATGATGAATCCGTAGATGTTTATGCTTTTGGAATGTGTATGCTGGAAATGGCCACATCGGAGTATCCTTATTCTGAGTGTCAGAATGCAGCTCAAATATACCGGAAAGTAACTAGTGGCATAAAACCAGCCAGCTTCAATAAAGTCACTGATCCTGAAGTCAAAGAAATCATTGAAGGATGTATTCGTCAAAACAAATCTGAAAGGTTGTCTATCAGGGACCTATTAAACCATGCATTTTTTGCTGAGGATACAGGACTGAGGGTGGAGTTAGCAGAAGAAGATGATTGCTCAAATTCATCCCTTGCTTTAAGACTCTGGGTTGAAGACCCTAAAAAATTGAAAGGCAAACACAAAGACAATGAAGCTATTGAATTTAGTTTCAACTTAGAAACAGATACACCTGAGGAAGTAGCATATGAAATGGTCAAGTCTGGGTTCTTCCATGAAAGTGATTCCAAAGCTGTTGCTAAATCCATTAGAGACCGGGTGACGCCAATAAAGAAGACAAGAGAGAAGAAGCCTGCTGGCTGTTTGGAAGAACGCAGGGATTCTCAGTGCAAGTCTATGGGGAATGTATTCCCTCAGCCCCAGAATACAACTTTACCCCTTGCTCCCGCTCAGCAAACTGGGGCTGAATGTGAAGAAACTGAAGTTGATCAACATGTTAGACAACAGCTTCTACAAAGAAAACCACAGCAGCACTGCTCCTCTGTTACAGGTGACAATTTGTCTGAGGCAGGAGCTGCATCAGTTATACATTCAGATACTTCAAGTCAGCCCAGTGTAGCCTATTCCTCAAATCAAACGATGGGCTCTCAAATGGTTTCTAATATCCCGCAGGCTGAAGTAAATGTTCCAGGGCAAATTTATTCATCTCAGCAACTAGTAGGACATTACCAGCAAGTTTCAGGGTTACAGAAGCATTCAAAGCTGACTCAGCCGCAGATTTTGCCTTTGGTTCAAGGTCAGTCCACTGTTTTACCTGTACATGTCCTTGGACCGACAGTTGTTTCACAACCCCAGGTTTCCCCATTAACTGTTCAGAAGGTCCCACAGATAAAGCCTGTATCCCAACCAGTTGGAGCTGAACAACAAGCAGCTCTTCTAAAACCAGATTTAGTTCGAAGCTTGAATCAAGATGTGGCAACTACGAAGGAAAACGTCAGTAGCCCTGATAACCCAAGTGGAAATGGCAAACAGGATCGGATCAAACAGAGAAGAGCTTCCTGTCCCCGACCAGAGAAGGGGACTAAATTTCAGCTTACCGTCCTTCAGGTATCAACCTCTGGAGATAACATGGTAGAGTGTCAGCTGGAGACACACAACAACAAGATGGTCACCTTCAAGTTTGATGTTGATGGTGATGCGCCAGAGGATATTGCAGACTATATGGTTGAAGATAACTTTGTGCTGGAAAGTGAGAAAGAAAAATTTGTAGAAGAATTGAGAGCTATTGTAGGTCAAGCCCAGGAGATCCTTCATGTCCACTTTGCCACAGAAAGAGCCACTGGAGTTGATTCTATTACTGTGGACTCCAACAGTAGCCAGACAGGGTCATCTGAACAAGTACAGATAAATTCTACATCTACTCAAACCAGCAATGAATCTGCTCCTCAGTCATCCCCAGTTGGTCGGTGGCGATTCTGTATCAATCAGACGATAAGAAACCGTGAGACTCAGTCTCCTCCTTCTCTTCAGCATTCCATGTCTGCGGTTCCTGGCCGACATCCACTTCCTAGTCCAAAAAACACAAGTAATAAGGAAATATCACGGGACACATTGCTCACTATAGAAAATAATCCATGCCACCGTGCACTTTTCACCTCCAAATCAGAACACAAGGATGTGGTTGATGGTAAGATTTCTGAATGTGCTAGTGTAGAAACCAAGCAGCCAGCTATACTTTATCAAGTGGAAGATAACAGGCAGATAATGGCACCAGTTACTAATAGTTCCAGTTACTCTACTACTTCAGTTCGTGCAGTTCCAGCTGAATGTGAGGGACTCACCAAACAAGCAAGCATATTCATACCTGTGTATCCATGTCACCAAACTGCCAGTCAGGCTGATGCACTTATGTCCCATCCTGGCGAATCAACTCAGACTTCTGGTAACTCTCTTACAACTCTGGCATTTGATCAAAAGCCTCAAACCTTATCAGTACAGCAGCCAGCTATGGATGCAGAGTTTATTTCTCAAGAAGGAGAAACTACAGTGAACACTGAAGCAAGTTCTCCTAAGACAGTCATTCCCACTCAGACCCCTGGCCTTGAACCAACTACCCTTCAACCCACTACTGTCCTGGAATCAGATGGAGAAAGACCTCCAAAACTGGAGTTTGCAGACAACCGAATTAAAACTCTGGATGAAAAATTAAGAAACTTGCTCTATCAGGAGCACAGCATCTCTAGCATCTATCCCGAGAGTCAGAAGGATACCCAAAGCATAGACTCTCCATTTTCTTCCTCTGCTGAAGATACCCTCTCCTGTCCAGTGACAGAAGTCATAGCCATCAGTCACTGTGGAATTAAAGATAGCCCTGTACAATCCCCTAATTTCCAACAGACAGGCTCTAAGCTTCTGTCCAATGTGGCTGCAAGTCAGCCTGCTAATATATCAGTGTTCAAAAGGGACCTGAATGTGATAACTTCTGTACCCAGCGAATTGTGTTTACATGAGATGTCCTCAGATGCTTCACTTCCAGGGGATCCAGAGGCCTATCCTGCTGCTGTGTCAAGCGGTGGAGCCATTCATCTGCAGACAGGAGTGGAAACAGAAGAGATGAGATCAGCAATTGCTCCTGATCCCATTCCTCTGACACGGGAGTCCACAGCTGATACTAGGGCTTTGAATAGATGTAAAGCGATGAGTGGATCATTTCAGCGGGGTCGGTTCCAGGTGATTACAATTCCTCAGCAGCAGTCAGCAAAAATGACATCTTTTGGAATAGAACACATATCAGTGTTCAGTGAGACAAACCATTCTAGTGAAGAAGCCTTTATTAAAACAGCAAAGTCTCAGTTGGTAGAAATAGAACCTGCCACACAAAATCCAAAAACTTCGTTTTCTTATGAGAAGTTACAAGCTCTTCAGGAAACCTGTAAAGAAAATAAAGGAGTTCCCAAACAAGGTGACAACTTCTTATCTTTCAGCGCAGCTTGTGAGACTGATGTATCTTCAGTGACCCCAGAAAAGGAATTTGAAGAAACTTCAGCCACAGGAAGTAGCATGCAGTCTGGATCTGAACTGTTGCTTAAAGAGAGAGAGATATTGACTGCTGGGAAACAGCCTAGCTCTGATAGTGAATTTTCAGCCAGTCTTGCTGGCAGTGGAAAGTCAGTGGCAAAGACTGGTCCAGAGAGTAATCAGTGCTTACCACACCACGAAGAACAAGCTTATGCTCAAACACAGAGTTCACTCTTCTATTCGCCATCTTCCCCAATGAGCAGTGATGATGAATCAGAAATAGAGGATGAGGACTTGAAGGTGGAGCTTCAAAGATTACGAGAAAAACACATTCAGGAGGTGGTAAATCTTCAAACCCAGCAGAATAAGGAGCTGCAGGAGCTCTATGAACGCCTTCGGTCAATTAAAGATAGCAAAACCCAATCTACTGAGATTCCTTTGCCACCTGCATCACCACGTCGACCAAGATCTTTCAAAAGCAAACTTCGAAGCCGCCCCCAGTCCTTGACACATGTGGACAATGGCATAGTTGCTACAGATCCACTGTGTGTGGAGAGTAATGCAGCATCATGCCAACAGTCTCCAGCCAGTAAAAAAGGGATGTTCACAGATGACTTACACAAGCTGGTGGATGACTGGACAAAGGAAGCAGTAGGAAATTCTCTTATTAAGCCAAGTTTAAACCAACTTAAACAAAGTCAACACAAACTAGAGACAGAAAACTGGAATAAAGTATCTGAAAATACTCCGTCTACTATGGGCTACACATCAACATGGATTTCTTCTCTGTCCCAAATCCGTGGAGCTGTCCCAACTTCCTTGCCACAAGGACTCTCACTCCCTTCATTTCCTGGGCCATTATCATCATATGGAATGCCTCACGTTTGTCAGTATAATGCTGTGGCGGGGGCGGGGTATCCAGTACAGTGGGTAGGAATTTCAGGAACAACACAACAATCTGTAGTAATTCCCGCCCAATCTGGGGGACCATTCCAGCCAGGGATGAATATGCAGGCATTTCCAACTTCATCAGTGCAGAATCCTGCCACAATCCCTCCTGGTCCTAAATGA
Protein Expression in Insect cells
Medium: Insect XpressTM- from Lonza (Catalog no. BE12-730Q)
Antibiotics: Penicillin/Streptomycin at 0.1%(v/v) for preventing contamination
Procedure: Sf9 cells grown to log phase at 2e6/ml, and co-infected with 3 ml WNK3B-c005 P2 virus /L, cells incubated at 27°C, shaking at 100 rpm in Infors shakers (with 25mm throw), cells harvested 72h post infection.
Baculoviruses amplified in 250ml glass Erlenmeyer flasks, 50ml Sf9 cells at 2e6/ml supplemented with 2% v/v foetal bovine serum, harvested 66-72h post infection, with supernatant collected by centrifugation at 900g, 4°C. (the pellet can be used for small scale purification)
Protein Purification
Procedure: Cells were lysed by sonication, 0.125% polyethyleneimine added, and lysates were clarified by centrifugation at 50,000G. The supernatant was then applied to a Nickel-NTA gravity column and washed and eluted with 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 10-300mM imidazole. Protein containing fractions were then treated with TEV protease overnight at 4 °C prior to gel filtration on an S200 gel filtration column using 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP as the running buffer. The protein was concentrated to 12.5 mg/ml using a spin concentrator.
Columns: Column 1: Ni2+ affinity; Column 2: S200 Gel Filtration
Concentration: 12.3 mg/ml
Mass-spec Verification: Intact mass correct. Mostly monophosphorylated with some unphosphorylated.
Structure Determination
Crystallization: Crystals were grown using the sitting drop vapor diffusion method at 20 ֯C. Crystals were grown in 150 nl drops consisting of 2:1 mother liquor (30% PEG Smear Medium -- 0.1M ADA pH 6.5 -- 0.2M lithium sulfate) to protein (12.3 mg/ml). 25% Glycerol was added and the crystal was then mounted and flash frozen in liquid nitrogen.
Data Collection: Data was collected at beamline I03 at 100K.
Data Processing: Data was processed to a resolution of approximately 2.7 Å using XDS and either xia2 or autoPROC, or DIALS.
