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1) Protein expression-differs slightly depending on the exact construct used.

Note: Remember to collect samples from cell growth pre- and post- induction so you can confirm protein expression by SDS-PAGE. Like calbindin D_9k, all the mutants run anomalously on a gel, as if they were 5kDa rather than 9kDa proteins. Expression of WT calbindin is induced at temperatures above 30°C. It was not rigorously tested to see if this is true for the calbindin multi-mutants, but just to be safe, all incubations prior to induction are performed @ 30°C. Detailed information on the various mutant constructs is listed on the Chazin web page: http://structbio.vanderbilt.edu/chazin/wisdom/protected/Plasmids.html.

For those mutants cloned into the Invitrogen pCR T7CT-TOPO background, protein expression was done using BL21(DE3) pLysS cells grown in 2xYT media containing 100 micrograms/mL AMP and 34 micrograms/mL CAP. After growing to OD₆₀₀ = 0.5-0.8, cells were induced with 0.5mM IPTG, and the temperature was raised from 30^oC to 37^oC. Four hours post-induction, cells were harvested by centrifugation at 5000 rpm for 15 min. Cell pellets (assuming 6L prep) divided among two 50mL falcon tubes and each tube's cells resuspended in ~45mL lysis buffer (20mM imidazole, 20mM NaCl, pH 7.0). Can freeze cells at -80^oC at this point if need be or continue on to Step 2.

For those mutants cloned into the pSV271 background, protein expression was done in an identical manner, except using BL21(DE3) cells grown in media containing 15 micrograms/mL KAN and induced with 1mM IPTG.

2) Protein isolation

Note: Remember to collect gel samples from total cell lysate, supernatant, and pellet for SDS-PAGE analysis.

Thaw cells from Step 1 (if relevant).

Add ~0.5mg/ml lysozyme and 1% NP-40 and 0.2mM PMSF to each tube, and let shake for 15 min. at 29^oC in incubator.

Freeze tubes in ethanol/dry ice mixture for ~ 5 min

Thaw (#1)

Freeze tubes in ethanol/dry ice mixture for ~5 min

Thaw (#2)

Freeze tubes in ethanol/dry ice mixture for ~5min

Thaw (#3)

Tubes should look very "snotty" at this point from the chromatin release, if not, add more freeze/thaw steps until they look like the cells have been lysed. Once "snotty", add 10mM MgCl₂ and 0.8mM PMSF to each tube.

Add DNAse I to each tube (just take a small dip out of bottle using spatula-half-a pencil eraser head size is plenty) and incubate at 37^oC for 20 minutes. Tubes should not be snotty, but very liquidy when finished. Stop DNAse I by adding 15mM EDTA.

Transfer to centrifuge tubes, and spin at 13,000 rpm for 25min.

Transfer supernatant to sterile, 50mL falcon tubes(s) for column purification, making solution 0.1mM PMSF. If you can't run the column steps right away, store solutions in -80^oC freezer.

3) Column Purification

First, run samples over DEAE Sepharose column (pgs. 58-61), then over Superdex 75 sizing column (see pages 64-73). Page numbers refer to Chris Bunick's notebook, which has the exact parameters and buffers.

DEAE Sepharose

Buffer A: 20 mM imidazole, 20 mM NaCl, 1 mM EDTA, pH 7.0.

Buffer B: 20 mM imidazole, 500 mM NaCl, 1 mM EDTA, pH 7.0.

Washed and equilibrated column according to "modified wash" procedure on page 58.

Thawed frozen (-80 ^oC) cell lysate solutions that had been syringe filtered through a 0.2 micron filter (see pages 46-47). Made solutions 0.2 mM in PMSF.

Loaded 66 mL of cell lysate into superloop, and ran over DEAE Sepharose column according to the procedure in the notebook on page 58. UV chart recordings are shown on pages 59 and 60.

Superdex 75

The retained fractions were pooled and concentrated to ~10 ml using ultrafree-15 centrifugal filter device with a 5000 Da molecular weight cut-off (MWCO). Washed membrane with 10 ml milli-Q water at 10 min., 1900 g. Then began concentrating ~42 ml down to 10 ml. Used the Beckman centrifuge, swinging bucket rotor, 15 ml sample placed in upper reservoir and run at 4 ^oC, 1900g ~ 3377 rpm.

Started cleaning Superdex 75 Hi Prep 16/60 column with 173 ml of 20% ethanol (1.44 cv). Then began equilibration against 20mM Tris, 150mM NaCl pH 8.0. Equilibrated in 514ml, or 4.28 Ccv.

Ran the sample over the column using the protocol on page 64. SDS-PAGE was used to check the contents of the fractions and fractions #25 and 26 contained the desired protein. The total sample required 8 runs, and each looked very similar and the protein came out in the same peaks.

Pooled all fractions #25 and #26 from runs 1-8 (yielded ~48ml total volume), and made 0-1mM PMSF.

Removed 20 mL for dialysis into chelex water following a 3-step process. First, dialyzed into 2L of 20mM Tris, 1mM EDTA, pH 7.8. Secondly, dialyzed into 2L of 10mM Tris, 1mM EDTA, pH 7.8. Both of these changes were at 4^oC. Thirdly, dialyzed into 2.2L of chelex water at 4^oC, pH 7.2. All buffers were made 0.1mM PMSF at start of dialysis.

Harvested ~23ml of solution from dialysis bag. Will begin to concentrate. Did two 30 min. spins for 1 min @ 16,000g spin in an ultrafree-15 centrifugal device with a 5000 MWCO. Harvested sample (~500 microliters), but put back in concentrator for 6 min. because appeared it would not be concentrated enough. Went down to ~300 microliters,spun once more for 1 min. @ 16,000g. Then performed BCA quantification, see page 63. This sample is 2.6x more concentrated than the last.