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Materials and Methods: LMNB1 (3HN9)
| Structure | LMNB1 |
|---|---|
| PDB Code | 3HN9 |
| Entry clone accession | MGC AT109-C2 (BC103723) |
| Tag | N-terminal tag: mhhhhhhssrenlyfqg |
| Construct sequence | MHHHHHHSSRENLYFQGSSVSISHSASATGNVCIEEIDVDGKFIRLKNTSEQDQPMGGWEMIRKIGDTSVSYKYTSRYVLKAGQTVTIWAANAGVTASPPTDLIWKNQNSWGTGEDVKVILKNSQGE EVAQRSTVFKTTI |
| Vector | pET28A-MHL |
| Expression host | BL21 (DE3)_V2R |
| Growth medium | TB |
| Growth method | A 250 mL flask containing LB (Sigma L7658) supplemented with 50 ug/ mL kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 ºC. Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 mL of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ mL kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 mL overnight LB culture, and incubated at 37 ºC. The temperature of the media was reduced to 15 ºC one hour prior to induction and induced at OD(600) = 6 with 100 uM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 ºC, and cell pellets collected by centrifugation and frozen at -80 ºC. |
| Extraction buffers | Lysis buffer: 20 mM Tris pH 7.5, 400 mM NaCl, 1mM phenylmethane-sulfonyl fluoride |
| Extraction procedure | Frozen cell pellet obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 200-250 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet. |
| Purification buffers | Wash Buffer: 20 mM Tris, pH 7.5, 400 mM NaCl, 25 mM imidazole Elution Buffer: 25 mM Tris, pH 7.5, 400 mM NaCl, 500 mM imidazole Gelfiltration Buffer: 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT |
| Purification procedure | IMAC: Unclarified lysate was mixed with 2-3 mL of Ni-NTA superflow Resin (Qiagen) per 200 mL lysate. The mixture was incubated with mixing for at least 90 minutes at 4 ºC. The mixture was than loaded onto an empty comLum (BioRad) and washed with 50 mL wash buffer. Samples were eluted from the resin by exposure to 3-5 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280 Gel filtration chromatography: An XK 26x65 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 1.0 mL/min. The dialyzed sample after concentration from the IMAC step (approx. 3 mL) was loaded onto the column at 1.0 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled. |
| Protein stock concentration | Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 15 mg/mL. |
| Mass spec | Diffraction quality crystals were grown using the following protocol: 30% PEG 4K, 0.2 M MgCl2, 0.1 M Tris-Cl, pH 8.5, vapor diffusion, hanging drop, temperature 291k. |
