Table of contents Bacterial over-expression of protein Cell Growth: Preparing frozen cell pellets for purification Harvesting by centrifugation: Resuspending cells Lysing cells Clarifying lysate FPLC purification Nickel Affinity column (preset) Desalting column (Manual) Concentrating and quantifying protein SDS-PAGE gels Crystallography screens Recipes Resolving X-Ray Data

Bacterial over-expression of protein

Cell Growth:

Mid-to-late afternoon, start a 10 mL overnight culture (luria broth with 100 ug/mL ampicillin). Inoculate using a single colony on a competent plate. Grow at 37 °C with shaking (~16 hours). The following morning, add 10 mL of overnight culture to 1 L Luria Broth/Amp containing growth flasks and grow at 37 °C in a shaker for three hours or until A600 = ~0.6 AU. Add 1.0 mL of 1 M IPTG to each 1.0 L flask, as well as ZnSO4 to 1 mM, and continue growing overnight.

Auto-lysing Alternative
If an auto-lysing strain of E. coli is available, follow the above instructions with a single colony from a competent plate, then add 40 mL of 0.3 M L(+)-Arabinose to the 1 L of broth in a growth flask along with the 10 mL overnight culture. Proceed as above, adjusting the amounts of IPTG and ZnSO4 to correspond to the increased volume (1 mM for both).

Preparing frozen cell pellets for purification

Harvesting by centrifugation:

Preweigh four 500-mL polycarbonate centrifuge bottles. Split your cultures roughly evenly across the bottles and balance prior to centrifugation. Centrifuge with the JA-10 rotor at 5000 x g for 15 minutes at 4 °C. Decant the spent media and weigh the cell pellet (record this number). Combine all the pellets into one falcon tube, label (including the actual mass of cells in the tube) and store in a -20 ˚C freezer until needed.

Resuspending cells

For each gram of cell pellet, add 3 mL of Buffer A (but no more than 40 mL). Add 4 μL of 100 mM PMSF and 80 μL of 10 mg/mL lysozyme per gram of cell pellet, plus a pinch of DNase. Transfer to an appropriately-sized beaker or falcon tube and stir, scrape or vortex until you’ve resuspended the pellet. Incubate for 20-30 minutes at room temperature while shaking to improve lysis by resuspending the pellets and allowing lysozyme time to work. It is not important to have a homogenously suspended pellet because the lysing process will take care of any chunks that exist (solution will look green).

-If an auto-lysing strain was used, lysozyme need not be added.

Lysing cells

Lysis by Bead Beater:

Fill the pre-chilled homogenization chamber of the BeadBeater half-full with 0.1 mm glass beads, and add resuspended cells. Top off the chamber with additional buffer A so that there will be no air space left when the cap is attached. Disrupt cells 15 seconds, followed by a 45 second rest period to re-chill the homogenization chamber contents. Repeat this disruption/rest cycle a total of 8 times for complete homogenization.

For Auto-Lysis Strains
L(+)-Arabanose Induced Lysis (freeze-thaw lysis protocol):

If the alternative, auto-lysing option under Endogenous Nickel Affinity Lab Protocol (Cell Growth) was used, the Bead Beater method can be skipped. Thawing from -20 ˚C and resuspension of the pellet with the incubation at room temperature in the previous step will allow for lysis and homogenization.

Clarifying lysate

Save a 500 μL aliquot of the crude extract. Centrifuge the remaining crude extract in 30 mL polypropylene centrifuge tubes at 20,000 x g for 20 minutes at 4 °C in the JA-20 rotor. While the centrifuge is spinning, spin the 500 μL aliquot at 14,000 rpm (spin hard) for 10 minutes in the tabletop centrifuge. Save the supernatant at -20 °C in the tray for your section (labeled Cell-Free fraction (CF) along with the protein, date and your initials). Add 500 μL of Buffer A to the pellet and resuspend by pipetting up and down. Save the resuspended pellet at -20 °C (labeled CF Pellet (CFP) along with the protein, date and your initials).
Once the centrifuge has stopped spinning, decant the supernatant into a suitably sized graduated cylinder and record the volume of the supernatant. It may be necessary to filter the lysate through a 0.45 um syringe filter in order to avoid getting glass beads into the FPLC, however getting some cellular debris in the FPLC is permitted.

FPLC purification

Nickel Affinity column (preset)

The purification of overexpressed soluble proteins from E. coli lysis cultures requires the selective and reversible association of the protein of interest on a solid matrix containing some affinity characteristic. In our lab we are currently using engineered or endogenous histidine character of our enzymes which efficiently ligates divalent metals complexed to a column matrix. Competing concentrations of histidines functional group, imidazole, effectively elutes the protein from the ligation sphere of the metal, purifying it up to 95% in a single column.

You should be trained on the FPLC before using the machine.

A typical protocol follows:

* Clarified cell lysate from 1L of cells is loaded onto a 5 mL Ni-affinity column preequilibrated with 10-30 mM imidazole.
* The column is then washed with 3 column volumes (CV) of 10-50 mM imidazole to remove any loosely associated proteins. Additional washing may be needed if the A280 signal has not returned to baseline.
* A gradient of 10-500 mM imidazole over 5 CV is used to specifically elute the his-tagged proteins. Absorbance at 280 nm is monitored throughout the purification to track protein elution from the column.

Project-Specific Protocol:
1. Use a syringe body to purge the Superloop with Buffer A, then load the supernatant from the final centrifugation step (should be in Buffer A) between the stoppers of the Superloop.

• A forceful initial loading motion prevents the lysate from going through the bypass function. Make sure to remove air bubbles from the Superloop as you finish loading.

2. Attach the appropriate Ni-affinity column and use the HisTrap template on the small FPLC in HU112 to run the appropriate wash, then a gradient of Buffer B. Set your volume as read from the Superloop and collect the 1-mL fractions in 2-mL disposable tubes.

• As the chromatograph shows protein coming off, record the fraction number.
• After the full run, combine fractions that contain protein and export the absorbance, %B and fraction curves as Excel files.

Desalting column (Manual)

Pooled fractions from the Nickel Affinity column are run over a desalting column in order to remove excess imidazole and further purify the protein of interest.

1. Purge the Superloop with Desalting Buffer (Final buffer) then load the pooled fractions from the previous column.
2. Attach the 12 mL desalting column and wash with desalting buffer till the baseline flattens (1-2 column volumes)

• Manual run: flow rate = 5 mL/min, set to load (with the lead in the desalting buffer).

3. Load the protein onto the column and run it through, collecting 1 mL fractions.

• Manual run: flow rate = 1 mL/min, fraction size 1 mL, set to inject.

4. Wash the column (see 2 above).

If the desalting column is not available:
Save the FPLC aliquots in 3x desalting buffer overnight in order to avoid precipitation. The next day centrifuge using a molecular weight filter at 3500 RPM until solvent goes through the filter - save the eluent and the filtrate to run in SDS-PAGE.

Concentrating and quantifying protein

Concentrating For SDS-PAGE and Crystal Set-up
The pooled fractions from FPLC should be run through a concentrator into a 50-mL falcon tube by loading the top up to 8 mL and centrifuging at 4,000 rpm for 10 minutes. The tube can be topped off to approximately 8 mL again, then spun again, repeating then spinning down to a final volume ~2 mL and discarding flow-through as necessary. Final solution should be quantified via absorbance (see below).

• Confirmation of purity and appropriate protein size may be obtained by running a denaturing PAGE gel with appropriately diluted samples. Protein may then be used experimentally or for use in crystallography set-ups.

SDS-PAGE gels

The protocol for SDS-PAGE will be discussed during instrument training. You should, by the end of the purification, have several samples to run on the gel: Cell-Free Fraction, Cell-Free Pellet, Nickel Affinity Fraction and Desalting Fraction. You will also want to run at least one lane of molecular weight markers.

Quantifying Protein

You will also want to quantify protein concentration in each of the samples listed above. In addition to quantifying total protein concentration using the BCA Assay Kit (see kit for instructions), you should take a total absorbance spectrum of the sample to confirm pure protein without nucleotide contamination.

An alternative method to using the BCA Assay is to take an absorbance reading at 280 nm of your purified protein solution, diluted down to yield an accurate reading. The extinction coefficient and molecular weight for the protein can be found through ExPASy Protparam, and the concentration of the solution can be found, then used to approximate the amount (mg) of protein present

Crystallography screens

A crystal screen is available in the lab for hanging drop set-ups. If your protein was purified via nickel column chromatography, use a concentrator to achieve a final concentration of approximately 10 mg/mL (record the net mg yield or concentration and volume). Then, set up 1ul x 1ul drops over 0.5 mL of well solution for the first 24 solutions in the screen. Observe your tray 24-48 hours later, and if more than 1/3 of the drops are visibly opaque (have precipitated) set up the next 24 solutions using 0.25 mL of the screen solution and 0.25 mL water in the well. Check your drops under a microscope for any crystal hits, and fine screen to optimize conditions if time allows. Diffraction quality crystals should be cryo-frozen and stored under liquid nitrogen. Take photographs of crystals to document on the wiki (record all the relevant conditions of the hit.)

Four crystal trays were set up using a concentrated sample of the HICA (6 mL) that was run through a sizing column. The sample was concentrated to a centrifuged to a concentration of 8.28 mg/mL in a Viva Spin tube and two centrifuge tube-sized concentrators at 4000 rpm and 10,000x g, respectively. The trays were set up in the following manner:

• Crystal Screen Solutions 1-24, pure well solution
• Crystal Screen Solutions 25-46, pure well solution
• Crystal Screen Solutions 1-24, 50% well solution, 50% ultra-pure water
• Crystal Screen Solutions 25-46, 50% well solution, 50% ultra-pure water

(Edited by D. Lee 3/19/13)

Recipes

Solutions Needed:

Detailed notes about making the following solutions can be found in recipes

1. Luria Broth (LB): 1 L /protein pellet you wish to make (or you can use TB Medium, seen below) plus 10 mL per overnight culture

• To 1 L of deionized water, add 10 g tryptone, 5 g yeast extract and 10 g of sodium chloride. Do not worry about stirring; sterilization will dissolve the solids. Sterilize by autoclaving. Before use with bacterial cultures (the flask must be cool enough to touch), add ampicillin while stirring to a final concentration of 100 μg/mL. Make sure the sterile media remains sealed with aluminum foil as much as possible.

2. TB Overexpression Medium (See under notes): For 1 L,

• Nutrient Solution: add 12 g Tryptone, 24 g yeast extract, 8 mL 50% glycerol, make solution to 900 mL with DI water, and autoclave
• Buffer Solution: 2.31 g KH2PO4 (Monobasic), and 12.54 g K2HPO4 (Dibasic), make solution to 100 mL with DI water, and autoclave
• Do not mix solutions until immediately before use, at which point add whatever antibiotics necessary

3. Buffer A: 1 L total, 10 mM imidazole (optimal for ECCA), 20 mM Tris-HCL, 0.1 M NaCl, pH 8.0

• If no Tris-HCL, use 20 mM Tris and adjust pH using HCL
• For HICA and its mutants, up to 40 mM imidazole is suggested to get optimum purification.

4. Buffer B: 1 L total, 0.5 M imidazole, 20 mM Tris-HCL, 0.1 M NaCl, pH 8.0

• If no Tris-HCL, use 20 mM Tris and adjust pH using HCL

5. Desalting Buffer: 1 L total, 20 mM Tris-HCL, 0.1 M NaCl, pH 8.0

• If no Tris-HCL, use 20 mM Tris and adjust pH using HCL

6. (Optional) 0.3 M L-arabanose

Tris-Glycine SDS Running Buffer Catalog No. LC2675 (10x) 500ml Catalog No. LC26754 (10x) 4X1L
Tris Base (29 g) Glycine (144 g) SDS (10 g) Distilled water to 1 liter NOTE: 1x running buffer should be ~pH 8.3. Do not use acid or base to adjust pH.

"Crystallography"

Resolving X-Ray Data

1. Run CCP4 program
a. Import Scaled Data from D*trek file
b. Run program "pointless"
c. Run program "sortmtz"
d. Run truncate
e. Under Molecular replacment Menu, run Phaser CCA
f. Find a model protein in the PDB, download as text file into project folder. Run phaserMR and select text file as reference.
Actually, Hoffmann made us a hica-chainDF.pdb on the computer to use
g. Double click on output file and find the Fast Translation Z-score.

In-Phaser Z-scores:
Lysh: 1101.62
Greg: 969.791

Using the output pdb file, run Refmac7 to refine the model for the actual data. Use Coot to see whether the model and the data fit well.

Sort out the metal thing. Remove disulfide bonds across the zinc density.

Rerefine. Note the R-free and R-work--these are your error values, and they should go down as you refine, and stay within 8% of each other.