Entry Clone Source: Synthetic |
Entry Clone Accession: n/a |
SGC Construct ID: PB1A-c023 |
GenBank GI number: gi|30794372 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGCAGCTGTATGATACCGTG
CGTAGCTGCCGCAACAACCAGGGCCAGCTG
ATTGCGGAACCGTTCTATCATCTGCCGAGC
AAAAAAAAATATCCGGATTATTATCAGCAG
ATTAAAATGCCGATCAGCCTGCAGCAGATT
CGCACCAAACTGAAAAATCAGGAATATGAA
ACCCTGGATCACCTGGAATGCGATCTGAAC
CTGATGTTTGAAAACGCGAAACGTTATAAC
GTGCCGAATAGCGCGATCTATAAACGTGTT
CTGAAACTGCAGCAGGTTATGCAGGCCAAA
AAAAAAGAACTGGCGCGTCGCGATGATATT
GAATGACAGTAAAGGTGGATACGGATCCGA
A
|
Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smQLYDTV
RSCRNNQGQLIAEPFYHLPSKKKYPDYYQQ
IKMPISLQQIRTKLKNQEYETLDHLECDLN
LMFENAKRYNVPNSAIYKRVLKLQQVMQAK
KKELARRDDIE
^ TEV cleave site |
Tags and additions: Cleavable N-terminal His6 tag. |
Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain) |
Growth medium, induction protocol: 5 ml from a 50 ml overnight culture containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol were used to inoculate each of two 1 litre cultures of LB containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol. Cultures were grown at 37 °C until the OD600 reached ~0.5 then the temperature was adjusted to 18 °C. Expression was induced overnight using 0.5 mM IPTG at an OD600 of 0.9. The cells were collected by centrifugation and the pellet re-suspended in binding buffer and frozen. Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5 % glycerol. |
Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5 mM TCEP, 1 mM PMSF added to the lysate. Cells were lysed using sonication. The lysate was centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification. |
Column 1: Ni-affinity. Ni-sepharose (Amersham), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer. |
Column 1 Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% Glycerol; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol (step elution). |
Column 1 Procedure: The flow-through from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 30 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted. |
Column 2:Size Exclusion Chromatography. Superdex S200 16/60 HiLoad |
Column 2 Buffers: 25 mM HEPES, pH 7.5; 300 mM NaCl, 0.5 mM TCEP |
Column 2 Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 300 mM NaCl, 0.5 mM TCEP using an ÄKTAexpress system. |
Mass spec characterization: LC- ESI -MS TOF gave a measured mass of 15593 for this construct as predicted from the sequence of this protein. |
Protein concentration:Protein was concentrated to 9.3 mg/ml using an Amicon 3 kDa cut-off concentrator. |
Crystallization: Crystals were grown at 4 °C in 300 nl sitting drops from a 1:1 ratio of protein to reservoir solution containing 0.1 M NaOAc.3H2O; 1.1 M (NH4)2SO4. |
Data Collection: Crystals were cryo-protected using the well solution supplemented with 2M Li2SO4 and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected from a single crystal on Diamond beamline IO2 at a single wavelength of 0.9795 Å and the structure was refined to 2.2 Å. Phasing: The structure was solved by molecular replacement using an ensemble of known bromodomain structures as a starting model. |