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Resuspension of cell pellets

The extraction of overexpressed soluble proteins from E. coli cultures requires removal of the culture medium, breakage of cells, and clarification of the extract. All operations should be carried out at 4 °C or with samples placed on ice to minimize protein denaturation and proteoloysis.A procedure to create a cell pellet from overexpressed cells follows:

  • In 500 mL centrifuge bottles, pellet cells by centrifugation at 8000 xg for 10 min, discarding the supernatant
  • Transfer the cell pellets to tared, labeled 50 mL falcon tubes. Weigh the bottles to determine the mass of wet, pelleted cells.
  • Freeze overnight (at least overnight) in a -20 or -80 degree freezer. Flash cooling in liquid nitrogen is also effective.


For cell breakage we are currently using autolytic cells (Zymo Corp) which efficiently disrupts cells by growing them in the presence of arabinose-induced endolysin. A freeze-thaw cycle releases endolysin from a portion of the cells after harvesting, and the extracellular endolysin efficiently breaks the remaining cell walls to 90% efficiency over 20 minutes. A typical protocol follows:

  • Remove tubes from freezer and resuspend cells in approximately 3 mL/g cell pellet of extraction buffer by scraping, vortexing, and/or shaking vigorously. Add protease inhibitors, DNAse I, and lysozyme (optional). Shake until mixture is homogeneous +20-30 minutes.
    • One protease inhibitor mini tablet (may want to crush up tablet and/or dissolve in buffer)
    • 100 uL of protease inhibitor solution thawed (= enough for 20 g cells)
  • Balance the required number of 30 mL centrifuge tubes, and centrifuge at 35000 xg (or ultracentrifuge max) for 30 min to clarify the cell extract. Spin for another 15-20 min if solution is not clarified.
  • Carefully decant the supernatant for purification.(see below)

Notes

  1. It may be necessary to filter the supernatant through a 0.45 um syringe filter prior to FPLC purification to prevent any unclarified solids from damaging the instrument. It is also appropriate to spin the lysate a second time to ensure clarity.

BeadBeater to lyse cells

If the autolysis is incomplete or taking too much time in a long day, another option is to use a BeadBeater, which is something like a blender using glass beads to disrupt the cell membrane. All operations should be carried out at 4 °C or with samples placed on ice to minimize protein denaturation and proteoloysis. A typical protocol follows:

  • Resuspend cells in no more than 40 mL of extraction buffer by scraping, vortexing, and/or shaking vigorously.
  • Combine resuspended cells in one container, and add any protease inhibitors desired.
  • Fill the pre-chilled homogenzation chamber of the BeadBeater™ half-full with 0.1 mm glass beads (just cover the blades of the agitator), and add resuspended cells.
  • Top off the chamber with additional extraction buffer 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.
  • Load the crude cell extract into the required number of appropriately balanced 50 mL centrifuge tubes.
  • Centrifuge at 48000 xg for 40-60 min to clarify the cell extract.
  • Carefully decant the supernatant for purification.


Notes

  1. The chamber of the Bead Beater can accommodate about 40-45 mL of cell suspension when properly filled with glass beads.
  2. It is critical that there is no air in the homogenization chamber. Air in the chamber will reduce disruption efficiency and may denature extracted protein.
  3. It may be necessary to filter the supernatant through a 0.45 μm syringe filter prior to FPLC purification to prevent the tiny glass beads from damaging the instrument.


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Purifying proteins via FPLC column chromatography

Cleaning and Priming

Most "problems" with the FPLC may be traced back to either air in the lines, or solids (salts, insoluble cell material) clogging a valve or column.

A general cleaning procedure before any run might include the following:

  1. a quick rinse of the system lines using ethanol and/or filtered, visibly clear water (it is a good habit to always check that media are appropriately clear). The system, provided the position1bypass is being used, may be run up to 10 mL/min on a 50% gradient to flush both A and B lines. This is always indicated after FPLC use, and may be a good idea if the previous run was left in a less than hygenic state.
  2. priming the pumps by withdrawing ~15 mL of material from pumps A and B (usually A is the culprit)--this is especially indicated if there is less volume flow than expected.

Cleaning and maintenance post-run.

  1. One's buffer should not be left in the FPLC lines. The system should be rinsed with filtered magic water, and then ethanol at the end of the summer (do not follow a glycerol buffer directly with ethanol.) This is a good opportunity to also rinse the syringes used for loading the loop.
  2. A measure of filtered di water should be loaded back into the superloop, to clear the line between the loop and the valve.
  3. All used fraction tubes, waste liquid, products and lysate containers, should be disposed of or stored, as relevant.

Methods

Individual labs using the AKTA FPLC consistently are encouraged to program a method for the researcher's use (frequently named for the lab, then the column, i.e. Hoffmann24mLGEC). If the lab has their own columns (encouraged unless cost is prohibitive) they may leave their column permanently connected to a free column position.

Affinity columns

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.
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.
  • 1 mL fractions are collected for the duration of the gradient, and fractions corresponding to the peak of interest are immediately pooled and passed over a 15 mL desalting column to remove imidazole. Alternatively, a rough buffer exchange may be achieved using Vivaspin concentrators and selective dilution with low-salt buffer, or a classic overnight dialysis may be performed.
  • Confirmation of purity and appropriate protein size may be obtained by running a denaturing PAGE gel. Protein may then be used experimentally or concentrated for use in crystallography set-ups.

Notes

  1. It is critical that all buffers and lysates are filtered before they are pumped through the FPLC. It is a very good idea to habitually visually inspect whether anything has started growing in your media.
  2. Histidines ligate Ni2+ very efficiently; if a protein of interest fails to elute with 0.5-1M imidazole, a cobalt affinity or other metal affinity column might change the ligation conditions to more favorably reverse the interaction with the tagged protein.
  3. Increasing the amount of imidazole in the lysis buffer and wash buffers will decrease the number of contaminating proteins in the prep, but will also lower the amount of imidazole required in the gradient to specifically elute the tagged protein.
  4. Proteins are often less soluble in high amounts of imidazole. Buffer exchange via desalting column, dialysis or dilution/concentration is recommended immediately after eluting from the nickel column. Additional additives in the buffer, such as 5-10% glycerol, may help with solubility in imidazole.

 

Gel-exclusion Chromatography (GEC)

Also known as a sizing column, GEC is simultaneously a purification step, buffer exchange, and analysis of quaternary structure (size) in solution. The separation is effective due to the porous nature of the gel matrix, allowing proteins of large size to bypass most of the crevasses and elute first (a size large enough to bypass all the crevasses is not going to be separated from other things of large size, and elutes in the void volume, or the edge of separation.) Proteins, small molecules and ions take a much longer path through the column as they detour into any nook and cranny they can fit into, thereby eluting slower based on size, with salts and buffers being completely separated from proteins, unless they are in the column buffer.
In a typical run:

  • The column is washed with 3-4 column volumes (CV) of the desired final buffer. No secondary buffer is required.
  • the concentrated protein solution is applied in volume no more than 1/100 of the column bed volume. For our smaller sizing columns at 24 mL, we try to load no more than 250 uL. *Immediately after applying the protein, or at latest at the void volume, begin collecting fractions.
  • pool desired fractions and concentrate for use in experiments
  • wash the sizing column with water between runs, and with 0.1 M NaOH if the backpressure starts to creep up (proteins or salts may have precipitated in the column matrix.)
  • Using the chromatograph, estimate the elution volume (Ve) in mL, and use the standardized spreadsheet for the column to accurately estimate molecular weight of the biological complex in solution. You will need to use the same amount of glycerol in solution.

Notes

  1. Sizing columns are run at about 0.5 ml/min or less to maximize separations. If you need better resolution between protein peaks, run slower or apply your protein in a smaller volume to the column.
  2. calculating based on elution volume, not time, means the spreadsheet is independent of the rate used to run the column.
  3. The spreadsheet for the 24 mL Superdex 200 increase column in 5% glycerol is here: GEC-increase-Jan2014
  4. The spreadsheet for the 60 mL manually-poured column in 5% glycerol is here: Big-SEC calibration

Using Excel to make a chromatography figure

There are a number of good programs available to help make figures of chromatography runs (such as GraphPad Prism, which has a free trial for 30 days) but Excel is used throughout many curriculums and is standard practice at Gonzaga. The default chart in Excel is also supremely distracting to look at. The following commands were noted in the Mac excel interface, but adaptations to the PC can probably be found by choosing a search term carefully in the help menu ("adding secondary axis to a chart" for instance.)

To format a clear, readable chart in Excel (Mac interface):

  • choose the chart tab and make a marked scatterplot of appropriate data (do not include any lines unless they are calculated fit lines),
  • then choose chart layout (the chart must be selected) and the gridlines pulldown menu. Under the (primary) horizontal lines option, select no gridlines to clean up the background of the graph.
  • Many journals (and professors) will ask you to close the axes for looks and layout purposes (and to keep the figure from looking like data are flying off into space.) To do this, make sure the axes are at the edges of the figure. Then double click on the center of the chart to pull up the format plot area menu. Under line, select black. If you have multiple data series on the same graph (see below), you can also select "axis"> secondary horizontal axis > axis without labels to do the same thing.
  • If you have multiple data series on the same chart (the A280 trace and the gradient, for example), you must include both axes. To add a secondary axis to the right hand vertical axis of the chart, you need to already have the data plotted on the chart(you can add to an existing chart using the very top "chart" pulldown menu and "add data".) Highlight the data you want to plot on the secondary axis (click a data point, for instance,) then, in chart layout, on the far left side of the menu is "current selection" Click format selection. In the navigation plane, pull down the axis menu, and click secondary axis. The chart layout navigation plane then has options to add an axis title ("axis title" > "secondary vertical axis title") and/or change the axis labels ("axis" > "secondard vertical axis" > labels).

removal of purification tags

Many proteins are conveniently expressed with purification tags, e.g., 6x His-tags, maltose binding protein, glutathione-S-transferase, etc. to simplify purification to homogeneity and/or to improve expression levels of soluble protein. These tags often must be removed before the protein of interest can be studied. One of the most common removal methods involves proteolytic cleavage of the purification tag utilizing a cloned protease (thrombin, TEV or similar) recognition site.

A typical protocol for thrombin cleavage of a tagged protein using a Novex Thrombin Cleavage Capture Kit follows:

  • In a 2.0 mL microcentrifuge tube add
    • ~2 mg tagged protein (up to 1600 uL)
    • 200 uL 10x thrombin cleavage buffer
    • water to a total volume of 1800 uL
  • Add 200 uL diluted biotinylated thrombin1 and mix gently by inversion
  • Incubate at room temperature for 2-24 hours2
  • Remove 2-5 uL for later SDS-PAGE analysis
  • Thoroughly resuspend streptavidin agarose slurry by inversion and pipet 32 uL into reaction mixture with a wide-bore pipet tip3
  • Incubate for 30 min at room temperature. Mix gently by inversion every few minutes to keep streptavidin agarose resuspended
  • Transfer no more than 350 uL at a time of the reaction mixture to the sample cup of a spin filter
  • Centrifuge at 500 xg for 30 s on a tabletop centrifuge. Remove filtrate with a transfer pipet to a clean 2.0 mL microcentrifuge tube
  • Repeat the previous two steps until all of the reaction mixture is processed, taking care to save the filtrate solution, which is your cleaved protein
  • Store protein at 4 ºC and purify protein as soon as practical using gel exclusion chromatography

Notes

  1. The exact dilution of thrombin should be determined by prior cleavage trials, and could vary from 1:20 to 1:400 dilution. To minimize non-specific cleavage, the greatest dilution of thrombin should be used that gives complete cleavage in the desired time interval.
  2. The exact incubation time should be determined by prior cleavage trials. Utilize the shortest cleavage time that results in complete cleavage using the desired thrombin dilution.
  3. In the absence of wide-bore pipet tips, cut off a portion of the end of a standard pipet tip to accommodate passage of streptavidin agarose particles

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