PTPTN5
PDB:2BIJ
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession: gi 22095375
Entry Clone Source:Origine
SGC Clone Accession:Tag:N-terminal His-tag with integrated TEV protease site: MHHHHHHSSGVDLGTENLYFQ*SH
Host:BL21 (DE3)
Construct
Prelude:Sequence:mhhhhhhssgvdlgtenlyfqsmSRVLQAEELHEKALDPFLLQAEFFEIPMNFVDPKEYD IPGLVRKNRYKTILPNPHSRVCLTSPDPDDPLSSYINANYIRGYGGEEKVYIATQGPIVS TVADFWRMVWQEHTPIIVMITNIEEMNEKCTEYWPEEQVAYDGVEITVQKVIHTEDYRLR LISLKSGTEERGLKHYWFTSWPDQKTPDRAPPLLHLVREVEEAAQQEGPHCAPIIVHCSA GIGRTGCFIATSICCQQLRQEGVVDILKTTCQLRQDRGGMIQTCEQYQFVHHVMSLYEKQ LSHQS
Vector:pLIC-SGC
Growth
Medium:Antibiotics:Procedure:Grow starter cultures from freshly transformed colonies in 10 ml LB , 0.1 mg/mL amp. This started culture was diluted 1:1000 in fresh media and was grown at 37oC to a OD600 of 0.3 and than transferred to 18 oC. Expression was induced at an OD600 of 0.8 using 1 mM IPTG. Cells were harvested after 3h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen.
Purification
ProcedureNi affinity, HisTrap (1 ml), in AKTA-express.
Loading buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, 5% glycerol. Wash buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 50 mM imidazole, 0.5 mM TCEP, 5% glycerol.Elution buffer: 50 mM Hepesl, pH 7.5, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP, 5% glycerol. Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of Loading buffer, 10 volumes of wash buffer, then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.
SEC: The peak collected from IMAC was directly applied to a S75 column equilibrated in 50 mM Hepes pH 7.5, 500 m NaCl, 5% glycerol.
Procedure: Akta Express
HiTrap Q: IEX buffer A: 50 mM Hepes, pH 7.5, 10% glycerol. IEX buffer B: 50 mM Hepes, pH 7.5, 1.0 M NaCl.
Protein fraction from desalting column loaded at 1 ml/min, the column was then washed with 10 ml of buffer A and eluted with a 20-minute gradient to 50% buffer B, followed by a step to 100% buffer B.
Concentration: Centricons 10 kDa cut off
Extraction
ProcedureExtraction buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM PMSF, 0.5 mM TCEP. The cell pellets (20 g wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 20,000 rpm in a JA 20 rotor
Concentration:LigandMassSpec:Crystallization:Crystals were grown at 4ºC in 200 nl sitting drops mixing 150 nl of PTPN5 (10 mg/mL in 50mM Hepes pH 7.5, 200mM NaCl ,10mM DTT) with 50 nl of a solution containing 25% PEG3350, 0.2M LiSO4, 100 mM Bis Tris Propane pH 5.5.
NMR Spectroscopy:Data Collection:Data Processing:
