Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:4081713 |
SGC Construct ID: AASDHPPTA-c003 |
GenBank GI number: gi|20357568 |
Vector: pCOEX-1 |
Tags and additions (His 6 + TEV cleavage site):
mgsshhhhhhhssgrenlyfqgh |
Host : E.coli BL21 DE3-R3 |
Construct coding sequence:
mgsshhhhhhhssgrenlyfqghMEGVRW
AFSCGTWLPSRAEWLLAVRSIQPEEKERI
GQFVFARDAKAAMAGRLMIRKLVAEKLNI
PWNHIRLQRTAKGKPVLAKDSSNPYPNFN
FNISHQGDYAVLAAEPELQVGIDIMKTSF
PGRGSIPEFFHIMKRKFTNKEWETIRSFK
DEWTQLDMFYRNWALKESFIKAIGVGLGF
ELQRLEFDLSPLNLDIGQVYKETRLFLDG
EEEKEWAFEESKIDEHHFVAVALRKPDGS
RHQDVPSQDDSKPTQRQFTILNFNDLMSS
AVPMTPEDPSFWDCFCFTEEIPIRNGTKS |
Growth medium, induction protocol: Medium: TB + 34 µg/ml Chloramp. Cells were grown in 1 liter TB in 2.5-L baffled flasks, which was inoculated with 10 ml overnight culture. The culture was grown at 37°C to OD=2.3, and transfered to 25°C. 1 mM IPTG was then added, and incubation continued for 4 hours. The cells were then collected by centrifugation and frozen at -80°C. |
Extraction buffer, extraction method: Lysis buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, Complete® protease inhibitors (1 tablet/50 ml). Frozen cell pellets were thawed on ice over night and resuspended in a total volume of 40 ml lysis buffer, the cells were disrupted by high pressure (20 psi) followed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI from a 5% (w/v) stock, stirring for 15 minutes, then centrifugation for 20 minutes at 40,000xg. The supernatant was then further clarified by filtration (0.45 µm). |
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham) |
Buffers: Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM Imidazole, 5% glycerol, 0.5 mM TCEP. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM Imidazole, 5% glycerol, 0.5 mM TCEP. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 30 mM Imidazole, 0.5 mM TCEP.
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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 Lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected. |
Column 2 : Gel filtration, Hiload 16/60 Superdex 200 prep grade 120 ml (GE Healthcare) |
Buffers : GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP. |
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. |
Concentration : In vivaspin 6 ml 10 K. Concentration determined from the absorbance at 280 nm using the extinction coefficient and the nanodrop method. |
Concentration : 19.6 mg/ml |
Enzymatic treatment : no cleavage |
Mass spectrometry: observed mass corresponds to predicted protein sequence |
Crystallization: 2BYD: Crystals were obtained using the following conditions: Vapour diffusion method, sitting drop, 293K, 0.05 M H3Cit/Na3Cit, pH 5.7, 14% PEG 3350. 2C43: Crystals were obtained from 2.0 M NaCl and 10% PEG6000 at 293 K. |
Data Collection: 2BYD: Resolution: 2.0Å, X-ray source: Synchrotron SLS -X10, multiple wavelength. 2C43: Resolution: 1.9Å, X-ray source: Rigaku FRE, single wavelength. |
