Overexpression of folded soluble protein

Pilot scale overexpression

The following protocol is convenient for preliminary assessment of overall protein overexpression in E. coli harboring vectors with trc or lac promoters and an ampicillin resistance gene.

• Using sterile technique, transfer 2 mL of LB medium containing 100 ug/mL ampicillin into a sterile 10 mL (17 x 100 mm) culture tube.
• Inoculate the medium with 100 uL of a fresh, overnight E. coli culture harboring the appropriate overexpression plasmid.
• Incubate with shaking (250 rpm) for 2½-3 hr. Take a 1 mL sample of cells, spin down gently and store at -20C for later PAGE analysis.
• Using sterile technique, add IPTG to a final concentration of 0.2-1.0 mM, to induce overexpression.
• Incubate with shaking (250 rpm) for 6 hr to overnight. Remove 1 ml aloquots at appropriate 1-2 hour intervals, spin down and store cell pellets frozen.
• Perform SDS-PAGE analysis of whole cells: resuspend the cell pellets collected in 30uL lysis buffer for 20 minutes. Spin down cells for 10 minutes at >11,000 rpm and collect supernatant. Combine with loading dye and run PAGE gels as described.

In order to determine what, if any, fraction of the overexpressed protein is produced in soluble form, it is necessary to grow a slightly larger culture in order to produce cell-free extracts. This is conveniently done immediately following the preliminary pilot overexpression.

• Using sterile technique, place 100 mL of LB medium containing 100 ug/mL ampicillin into a sterile 300 mL culture flask. (see below)
• Using sterile technique, pipet 1.0 mL of fresh, overnight E. coli culture into each of two (2) sterile 1.5 mL microcentrifuge tubes.
• Centrifuge at 6000 xg for 3 minutes, remove the supernatant, and resuspend in 1.0 mL of sterile growth medium.
• Centrifuge at 6000 xg for 3 minutes, remove the supernatant, and wash once again with 1.0 mL of sterile growth medium.
• Resuspend cells in each tube with 1.0 mL of sterile growth medium, and use the contents of both tubes (2.0 mL) to inoculate the 100 mL of growth medium previously prepared.
• Incubate with shaking (250 rpm) until A600 reaches 0.6-1.0, about 2½-3 hr.
• Using sterile technique, add IPTG to a final concentration of 0.2-1.0 mM, to induce overexpression.
• Incubate with shaking (250 rpm) for 6 hr to overnight.
• Harvest cells by centrifugation at 8000 xg for 5 min in two 50 mL centrifuge tubes.
• Wash cells twice by resuspension and centrifugation in cold 0.9% NaCl (see below)
• Take up wet cell pellet (typically 0.5-3 g) in 15 mL of the appropriate extraction buffer(see below) and freeze-thaw to lyse cells.
• Analyze expression via SDS-PAGE analysis.

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Production Scale overexpression

The large-scale overexpression of target proteins typically requires 1-4 L of cell culture. Such cultures cannot be started directly from single colonies on agar plates, but must be scaled up gradually. The following protocol is typical for most overexpression systems, and should observe proper sterile technique:

• The desired strain of E. coli harboring the desired overexpression plasmid should be streaked out on an agar plate containing the appropriate antibiotic and grown overnight.
• A single colony should be used to inoculate 10 mL of LB medium containing 100 ug/mL ampicillin or appropriate antibiotic, and the culture shaken overnight at 37 °C. Alternatively, cells can be grown until A600 reaches 0.6-1.0, and then stored at 4 ºC for up to 24 hr.
• For each 1 L of overexpression culture, place 10 mL of overnight scale-up culture in a 50 mL centrifuge tube.
• Pellet cells at 8000 xg for 5 min, and wash with 10 mL fresh LB medium with antibiotic as described above.
• Resuspend washed and pelleted cells in 10 mL of fresh LB medium.
• Prepare overexpression cultures by adding 1 L of TB medium containing the appropriate antibiotic to 2.5-3.0 L culture flasks.(see below)
• Inoculate each 1 L of overexpression medium with the entire contents (10 mL) of one 50 mL centrifuge tube.
• Incubate at 37 °C with vigorous shaking for 2½-3 hr, or until A600 reaches 0.6-1.0, then induce by adding IPTG to a final concentration of 0.2-1.0 mM.(see below)
• Grow cells 6 hr to overnight with vigorous shaking at 25-37 °C.(see below)
• Harvest cells immediately by centrifugation at 8000 xg in 500 mL centrifuge bottles, or store at 4 °C for no more than a few hours before processing. The typical yield of wet pelleted cells is 20-30 g per liter of culture for cultures grown at 37 ºC.
• If pelleted cells are not to be processed immediately, store at -80 °C.

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Optimized Overexpression for DesC

The following protocol has been shown to optimize the over-expression of DesC in the soluble fraction of the cell culture. Reference the DesC project notebook to see diagnostic protocol and SDSPage gel.

• Innoculate a 10mL volume of LB medium with a single colony of BL21 transformants.
• Grow overnight the 10mL volume of cells in a 15mL culture tube at 37°C
• Warm 1L of TB medium at 37°C and centrifuge the 10mL of cell culture at 2500rpm for 10minutes, discard the supernatant and resuspend cells in 2-4mL of TB medium.
• Add 100ug/mL ampicillin and 3mM arabinose to the TB medium and innoculate with the resuspended cells.
• Place Tunair flasks with cell culture in 37ºC at 215rpm until cell culture, when measured at 600nm in the UV spec, is between 0.5 and 0.6 absorbance units.
• Innoculate culture with 0.5mM IPTG
• Place cell culture back at 215rpm in 15ºC for 4-6 hours.

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Optimized Overexpression for DesD

The following protocol has been shown to optimize the over-expression of DesD in the soluble fraction of the cell culture. Reference the DesD project notebook to see diagnostic protocol and SDSPage gel.

Prep for overexpression:

Make 1n L of TB broth recipes and autoclave, prepare DesD plasmid molecular biology from DH5a cells (~1/semester) and transform XJb/BL21 cells molecular biology.

The day before overexpression:

• Innoculate a 10mL volume of LB w/ amp medium with a single colony of BL21 transformants.
• Incubate and shake O/N in a 15mL culture tube at 37°C

The day of overexpression:

• Warm 1L of TB medium at 37°C and centrifuge the 10mL of cell culture at 2500rpm for 10minutes, discard the supernatant and resuspend cells in 2mL of LB medium.
• Add ampicillin and arabinose to final concentrations of 100ug/mL and 3mM respectively to each liter of TB media recipes, remove 1 mL of TB medium to use as a blank for cell growth measurements, and then innoculate the 1 L sample of TB medium with the resuspended cells. Arabinose is a crucial additive that prepares autolytic cells for lysis. Your prep will go to hell if you do not add it.
• Place Tunair flasks with cell culture in 37ºC at 215rpm until cell culture, when measured at 600nm in the UV spec, is between 0.5 and 0.6 absorbance units (usually around 3-5 hours, often closer to 3)
• Innoculate culture with 0.5 mM IPTG
• Place cell culture back at 215rpm in 15ºC for overnight growth.

Optimized Overexpression for FslA

The following protocol outlines variations on the Production Scale Overexpression Protocol described above, which have demonstrated optimization of overexpression of fslA in the soluble fraction of the cell.

• Innoculate 5-6 mL of LB/Amp media with a single colony of BL21 transformants, and grow overnight (shaking) at 37 ºC
• Warm 1 L TB Media Broth to 37 ºC, while centrifuging 8-10 mL overnight cultures at 2,500 rpm for 10 minutes.
• Make the TB Broth to 100 ug/mL ampicillin and 3 mM arabinose, and save a 1 mL sample of the TB/amp/arabinose solution to use as a reference ABS blank.
• Discard the LB solution from the centrifuged cell cultures and resuspend the cell pellet in TB/amp/arbinose solution.
• Innoculate TB Media in sterile Tunair Flasks with resuspended cells.
• Place Tunair Flasks with cell cultures in 37 ºC incubation room and leave shaking at 215 rpm, until the cell culture, when measured at 600 nm in the UV spec, is between 0.6-0.8 ABS units relative to TB Broth ABS blank.
• Induce culture with 0.25 mM IPTG (500 uL of 0.5 M IPTG solution).
• Place induced cell culture(s) in 15 ºC environmental chamber and grow overnight.

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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)

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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 the in-line filter.

A general cleaning proceedure 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.
3. changing the inline filter for one that has been sonicated in isopropanol or NaOH (be sure to sonicate the dirty filter for at least one cycle once you remove it). This is probably the only fix you need if the system pressure is spiking, and should result in system pressures ~0.2 mPa.

Cleaning and maintenance post-run.

1. One's buffer should not be left in the FPLC lines. The system should be rinsed with filtered di-water, and then ethanol (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.

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.

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

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