Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:4139392 |
SGC Construct ID: DYRK2A-c022 |
GenBank GI number: gi|4503427 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Coding DNA sequence: |
Tags and additions: Cleavable N-terminal His6 tag. |
Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain) |
| Expressed protein sequence (tag sequence in lowercase): mhhhhhhssgvdlgtenlyfq^sMGKVKA TPMTPEQAMKQYMQKLTAFEHHEIFSYPE IYFLGLNAKKRQGMTGGPNNGGYDDDQGS YVQVPHDHVAYRYEVLKVIGKGSFGQVVK AYDHKVHQHVALKMVRNEKRFHRQAAEEI RILEHLRKQDKDNTMNVIHMLENFTFRNH ICMTFELLSMNLYELIKKNKFQGFSLPLV RKFAHSILQCLDALHKNRIIHCDLKPENI LLKQQGRSGIKVIDFGSSCYEHQRVYTYI QSRFYRAPEVILGARYGMPIDMWSLGCIL AELLTGYPLLPGEDEGDQLACMIELLGMP SQKLLDASKRAKNFVSSKGYPRYCTVTTL SDGSVVLNGGRSRRGKLRGPPESREWGNA LKGCDDPLFLDFLKQCLEWDPAVRMTPGQ ALRHPWLRRRLPKPPTGEKTSVKR ^ TEV cleave site |
Growth medium, induction protocol: 5 ml from a 50 ml overnight culture containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol were used to inoculate each of two 1 litre cultures of LB containing 50 μg/ml kanamycin and 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 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. |
Buffers: |
Procedure: The lysate supernatant 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 |
Buffers: 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP |
Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP using an ÄKTAexpress system. |
Mass spectrometry characterization: LC- ESI -MS TOF showed that the protein was herterogeneously phosphorylated at up to 4 sites in accordance with a mass of 49279 for this construct as predicted from the sequence of this protein. |
Protein concentration: Protein was concentrated to 7.3 mg/ml using an Amicon 10 kDa cut-off concentrator. |
Crystallization: Crystals were grown at 4°C in 300 nl sitting drops from a 2:1 ratio of protein to reservoir solution containing 0.2M Na(ac); 0.1 M cacodylate pH 6.5; 30% PEG 8K. |
Data Collection: Crystals were cryo-protected using the well solution supplemented with 2M Li2SO4 and flash frozen in liquid nitrogen. |