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Materials and Methods: HMGCS1 (2P8U)
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| Structure | HMGCS1: Human 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 |
|---|---|
| PDB Code | 2P8U |
| Ligands | Acetyl-CoA |
| Entry clone accession | IMAGE:2819708 |
| Entry clone source | MGC |
| SGC clone accession | HMGCS1A-c007 |
| Tag | N-terminal His-tag with TEV protease cleavage site (Tag sequence in lower case): |
| Construct sequence | mhhhhhhssgvdlgtenlyfqsMDVGIVA LEIYFPSQYVDQAELEKYDGVDAGKYTIG LGQAKMGFCTDREDINSLCMTVVQNLMER NNLSYDCIGRLEVGTETIIDKSKSVKTNL MQLFEESGNTD IEGIDTTNACYGGTAAVF NAVNWIESSSWDGRYALVVAGDIAVYATG NARPTGGVGAVALLIGPNAPLIFERGLRG THMQHAYDFYKPDMLSEYPIVDGKLSIQC YLSALDRCYSVYCKKIHAQWQK EGNDKDF TLNDFGFMIFHSPYCKLVQKSLARMLLND FLNDQNRDKNSIYSGLEAFGDVKLEDTYF DRDVEKAFMKASSELFSQKTKASLLVSNQ NGNMYTSSVYGSLASVLAQYSPQQLAGKR IGVF SYGSGLAATLYSLKVTQDATPGSAL DKITASLCDLKSRLDSRTGVAPDVFAENM KLREDTHHLVNYIPQGSIDSLFEGTWYLV RVDEKHRRTYARRP |
| Vector | pNIC28-Bsa4 |
| Expression host | E. coli strain: BL21(DE3)-R3 pRARE2 |
| Growth medium | TB |
| Growth method | 10 μL of a glycerol stock was inoculated into 5mL of TB medium (supplemented with 50 μg/mL Kanamycin, 34 μg/mL Chloramphenicol) and cultured at 37ºC o/n in a shaking incubator (275 rpm). Next day 0.75 mL of o/n culture was used to inoculate 1 litre of TB medium (6x) and grown at 37ºC with vigorous shaking (160 rpm) until the culture reaches an OD600 of 1.4. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.5 mM, and further cultivated for 16 hrs. Cells were harvested by centrifugation at 6500 rpm for 10 min, and the cell pellet was stored at -20ºC until further use. |
| Extraction buffers | Lysis buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM Imidazole , CompleteŽ protease inhibitors (Roche, 1 tbl/50 ml) |
| Extraction procedure | Frozen cell pellets were thawed and resuspended in a total volume of 30-40 mL of lysis buffer, and disrupted by using Avestin C-5 microfluidizer, and a supernatant containing the target protein was obtained by centrifugation at 21,000 (rpm) for 45 minutes . |
| Mass spec | Corresponds to theoretical mass, as determined by ESI-TOF MS. |
| Crystallization | Crystals were grown by vapor diffusion in sitting drops at 4ºC. Before setting up the experiment acetyl-CoA was added to the protein to a final concentration of 5 mM. A sitting drop consisting of 50 nl protein and 100 ηL well solution was equilibrated against well solution containing 25% PEG 3350, 0.2 M ammonium sulfate, 0.1 M BIS-TRIS pH 6.5. The crystal was mounted in a loop directly from the drop and flash-cooled in liquid nitrogen. |
