Molecular Biology
Entry Clone Accession:
Entry Clone Source: Tony Pawson
SGC Construct ID: WNK3B-c001
Protein Region: E123-A500
Vector: pFB-LIC-Bse
Tag: N-6HIS;N-TEV
Host: DH10Bac
Sequence (with tag(s)): MGHHHHHHSSGVDLGTENLYFQSMEKNEKEMEEEAEMKAVATSPSGRFLKFDIELGRGAFKTVYKGLDTETWVEVAWCELQDRKLTKAEQQRFKEEAEMLKGLQHPNIVRFYDSWESILKGKKCIVLVTELMTSGTLKTYLKRFKVMKPKVLRSWCRQILKGLQFLHTRTPPIIHRDLKCDNIFITGPTGSVKIGDLGLATLMRTSFAKSVIGTPEFMAPEMYEEHYDESVDVYAFGMCMLEMATSEYPYSECQNAAQIYRKVTSGIKPASFNKVTDPEVKEIIEGCIRQNKSERLSIRDLLNHAFFAEDTGLRVELAEEDDCSNSSLALRLWVEDPKKLKGKHKDNEAIEFSFNLETDTPEEVAYEMVKSGFFHESDSKAVAKSIRDRVTPIKKTREKKPA
Sequence after tag cleavage: SMEKNEKEMEEEAEMKAVATSPSGRFLKFDIELGRGAFKTVYKGLDTETWVEVAWCELQDRKLTKAEQQRFKEEAEMLKGLQHPNIVRFYDSWESILKGKKCIVLVTELMTSGTLKTYLKRFKVMKPKVLRSWCRQILKGLQFLHTRTPPIIHRDLKCDNIFITGPTGSVKIGDLGLATLMRTSFAKSVIGTPEFMAPEMYEEHYDESVDVYAFGMCMLEMATSEYPYSECQNAAQIYRKVTSGIKPASFNKVTDPEVKEIIEGCIRQNKSERLSIRDLLNHAFFAEDTGLRVELAEEDDCSNSSLALRLWVEDPKKLKGKHKDNEAIEFSFNLETDTPEEVAYEMVKSGFFHESDSKAVAKSIRDRVTPIKKTREKKPA
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 2ml of kinase WNK3B and 1ml of lambda phosphatase (NINIELA), 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, 300 mM NaCl, 0.5 mM TCEP as the running buffer.
Columns: Column 1: Ni-NTA; Column 2: S200 Gel Filtration
Concentration: 9.6 mg/ml
Mass-spec Verification: Intact mass correct
Structure Determination
Crystallization: Crystals were grown using the sitting drop vapor diffusion method at 4 ֯C. Crystals were grown in 400 nl drops consisting of 1:2 mother liquor (0.1M citrate pH 5.0 -- 18% PEG3350) to protein (9.6 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 2.4 Å using Imosflm and scaled/truncated with aimless.