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Much of cellular control over actin dynamics comes through regulation of actin filament initiation. At the molecular level, this is accomplished through a collection of cellular protein machines, called actin nucleation factors, which position actin monomers to initiate a new actin filament. The Arp2/3 complex is a principal actin nucleation factor used throughout the eukaryotic family tree. The budding yeast Saccharomyces cerevisiae has proven to be not only an excellent genetic platform for the study of the Arp2/3 complex, but also an excellent source for the purification of endogenous Arp2/3 complex. Here we describe a protocol for the preparation of endogenous Arp2/3 complex from wild type Saccharomyces cerevisiae. This protocol produces material suitable for biochemical study, and yields milligram quantities of purified Arp2/3 complex.
Plant, animal and fungal cells all make use of dynamic rearrangements of actin filaments to move and reshape themselves (1). In yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, these rearrangements have been intensely studied in both vesicle trafficking (2,3) and in cell division (4). Force generation needed during yeast clathrin dependent endocytosis requires precise control of the initiation of actin filaments by the Arp2/3 complex (3,2).
The Arp2/3 complex is composed of one copy each of seven polypeptides (5–7), all of which are needed for function (8,9). Two of these polypeptides are the actin related proteins Arp2 and Arp3, from which the complex derives its name. There are five additional subunits, with no homology to actin, known as ArpC1, ArpC2, ArpC3, ArpC4 and ArpC5. The complex is basally inhibited, but can be activated by a collection of ligands known as nucleation promoting factors (10,7). Nucleation promoting factors are able to integrate a broad range of cellular signals to activate Arp2/3 complex at specific places and times (11). These ligands must both trigger an activating conformational change in the complex (12–14), and deliver the actin monomers that become the first subunits in the nucleated filament (15–17). The activity of the Saccharomyces cerevisiae Arp2/3 complex may be assayed in vitro through the increase in fluorescence of pyrene labeled actin upon polymerization (18) or through the microscopic observation of fluorescent actin filaments assembling on beads (19).
Given the complexity of this multi-protein complex, it is most typically purified from endogenous sources. In addition to purification schemes that use only standard chromatography (20–25), endogenous Arp2/3 complex can be purified through the use of an affinity column with an immobilized ligand. Immobilized ligand columns were used to originally identify the complex (5). More recently, the use of affinity beads bearing the VCA domain of the nucleation promotion factor N-WASP has proven of broad utility. This method was first described for the purification of Arp2/3 complex from bovine brain (26), but the general strategy has since been adapted to the purification of Arp2/3 complex from Saccharomyces cerevisiae (27), from Acanthamoeba castellanii (28), and from Schizosaccharomyces pombe (29).
We describe here our version of a protocol to purify Arp2/3 complex from commercially available bakers' yeast (Saccharomyces cerevisiae). The described protocol is derived from published work (27), but has several added purification steps that improve the final purity of the complex when isolated from commercial baker's yeast. We describe the Arp2/3 complex purification at a scale where milligram quantities may be prepared. We hope that others may use this protocol to purify Arp2/3 complex from Saccharomyces cerevisiae, and as a template to develop new protocols for the purification of the complex from additional sources. The purification protocol typically requires four days of work, after completion of the two additional protocols. We recommend that two people be involved during the first two days of the purification, and a single person complete the third and fourth days of work.
All buffer and salt stocks are prepared using ultrapure water (>18 MOhm, using Millipore brand Milli-Q water purification system). Except where noted, all solutions are filtered through a 0.22 µm cellulose acetate membrane. Working buffers are prepared by dilution of buffer stocks into prechilled ultrapure water. Where specific sources are recommended, the manufacturer and part numbers are indicated.
Recommended flow rates and operating pressures are typical for a column in good condition.
This is a time intensive protocol and greatly benefits from having 2 people working together. Using commercially sourced yeast, one person can perform Section 3.1 (Preparing Yeast Suspension) with two 1 lb/454 g blocks of yeast in less than a day. Typically, we repeat the procedure several times over 1 – 2 days, producing enough frozen prepared yeast suspension for several preparations. Section 3.2 (Expression of GST N-WASP VCA in E. coli) can be performed by one person, and may be performed while the yeast suspension is being prepared. The described protocol prepares enough GST N-WASP VCA for multiple Arp2/3 complex preparations. Both Sections 3.1 and 3.2 must be completed before the Arp2/3 complex can be purified. The protocol for Arp2/3 complex purification takes four full days, which we break into four Sections here (3.3 – 3.6). Two people working in a team are needed during the first two days (Sections 3.3 and 3.4). Typically, these two days require 12 – 14 hour workdays at the scale of prep described here. Working as a team keeps the process manageable. The third and fourth days (Sections 3.5 and 3.6) can be completed by a single person.
The preparation described here removes any preservatives and stabilizers from the suspension, or any remaining media components if produced in house. Sections 3.3 – 3.6 yield approximately 1 mg of purified Arp2/3 from 150 g of yeast cells. The described preparation begins with ~300 g of yeast cells, but we typically prepare more than one preparations worth of cell suspension at a time. Two 1 lb/454 g blocks can be washed at one time using a centrifuge equipped with a six position, 1 L swinging bucket rotor. This protocol generates roughly >2 L of resuspended cells; ensure that sufficient liquid nitrogen and freezer space are available to complete the protocol.
On the first day of the protocol, yeast cell suspension is lysed using a cell extruder and clarified by high-speed centrifugation. These are the principle limiting steps in the entire protocol and available hardware will greatly influence the overall preparation scale. If the preparation changes in scale, use the given VCA column and SOURCE15Q column sizes to guide rescaling of the column sizes. Finally, if the prep is scaled up substantially, it may be impractical to increase the volume of dialysis buffer. In that case, additional buffer change steps may be used. As described, this protocol takes approximately 14 hours to complete, with a second person needed through the first 8 – 10 hours.
The second day of the protocol begins with the dialyzing samples from the first day. This day prepares the GST-VCA column while dialysis continues, and then performs the affinity column step twice. If the prep has been scaled up substantially, it may be necessary to add an additional dialysis buffer change step. As described, this protocol takes approximately 14 hours to complete, with a second person needed through the first 8 – 10 hours.
The third day of the protocol begins with the pooled GST-VCA elution fractions. The buffer is exchanged and Arp2/3 complex is further purified using SOURCE15Q ion exchange chromatography. A preparative gel filtration column is then run as an overnight step. A single person can perform desalting, ion exchange, SDS-PAGE analysis, and beginning the gel filtration column in 8 – 10 hours.
The fourth day of the protocol begins by assessing the overnight gel filtration results by SDS-PAGE. If necessary a second pass over gel filtration is used to complete the purification. Otherwise, the complex is concentrated, quantified and frozen. A single person can complete the fourth day in about five hours if the additional gel filtration step is not needed, and in about 10 hours if the additional gel filtration step is needed. Concentrating and freezing of the complex can be delayed until a fifth day.
1The wild type budding yeast (Saccharomyces cerevisiae) used in this protocol can be grown in the lab or acquired from commercial sources. We have had good success with both routes. This protocol can be performed using in house grown yeast from either shaker flasks (yeast grown to roughly OD600 of 1 – 2 in 18 L YPD is a good starting point for this protocol), or produced at high density using a fermentation system. Given the cost and time needed to produce this quantity of yeast, we recommend purchasing yeast from a commercial source. Dry yeast should not be used. 1 lb cakes of fresh yeast are commonly used in small commercial bakeries. The yeast has a shelf life of about one month, and thus most bakeries receive frequent shipments and are willing to part with one or two cakes at a price that is much less than the cost of media used to grow the yeast in house. Alternatively, one may be able to acquire a very fresh case of cakes directly from a distributor. This protocol has had good results with Red Star #05020 cake yeast, although washing, resuspending, freezing and storing at −80°C an entire 20 lb case is impractical.
2PMSF is toxic. Care should be used when preparing this stock. In particular, use of gloves, lab coats, protective eye wear and dust masks will reduce inhalation and cross contamination. One effective strategy is to purchase <100 g bottles, and prepare the entire bottle at one time trusting the manufactures provided weight. The working concentrations are high enough that small errors due to variability in the manufacturer's weight will not have a significant effect. The stock should be stored as 1 mL aliquots in 1.5 mL microfuge tubes at −20°C. Once the stocks have been frozen PMSF will crystallize out of solution. Warming in a room temperature beaker of water for a few minutes is usually sufficient to bring the PMSF back into solution. Agitation by inversion may be needed. When adding PMSF to a buffer, avoid splashing the stock and buffers onto gloves, skin and eyes.
3For this protocol, we use N-WASP VCA to capture Arp2/3 complex because it was used in the first reported version of this protocol. N-WASP VCA binds Arp2/3 complex more tightly than most VCAs. Also of note, the GST fusion to N-WASP VCA means that, in addition to binding to the target affinity column, the VCA will be a dimer. VCA dimers are important here as they increase the affinity towards Arp2/3 complex appreciably, and the protocol may not work with lower affinity VCA monomers. It is not clear whether VCA constructs will perform better than CA constructs, but we have used a VCA construct composed of amino acid residues 393–504. This is expressed from a modified pGEX 2T (GE) vector such that VCA has an N-terminal GST fusion, with a short linker and a thrombin cleavage site separating them.
4The quantity of GST N-WASP VCA used to prepare the affinity column is not a critical element. As the binding of Arp2/3 complex from solution is fairly inefficient, there should be an excess of GST-VCA present. Using our bacterial expression system, we use cells from 0.5 – 1 L of LB culture for each 100 g of yeast cells lysed or 300 g of cell suspension lysed (as prepared in Section 3.1). The DEAE column will bind nearly all of the VCA dimers from solution, and the glutathione sepharose should be overloaded with VCA dimer. By overloading this column, we minimize contaminants at the cost of yield. This saves additional purification steps, and thus greatly shortens the VCA purification.
We have found that Arp2/3 complex is never completely captured when working with beads suspended in batch. Thus, we save the flow through following separating beads from lysate after a binding step. The flow through is then reapplied to the beads a second time, and the purification repeated. We have found that additional Arp2/3 complex can be purified by additional cycles of capture from the lysate, but that the purity and amount captured decreases with each cycle. Typically, the second pass has only 30 – 80% of the Arp2/3 complex captured in the first pass, but shows only an incremental increase in contaminants. The third pass typically shows a similar decrease in quantity captured, but a substantial increase in contaminants. Thus, two cycles seems the best compromise in terms of yield, purity and time involved.
5The induction of proteins can be best seen in SDS-PAGE analysis if the samples are well matched. We have good results by collecting ~1.5 mL of culture and measuring its optical density at 600 nm (OD600). Calculate how much of this sample is needed to prepare a matched expression sample using the formula: volume = 0.15/OD600 * mL. Place that volume of culture in a 1.5 mL tube, and pellet the cells by spinning at ~10,000 × g for 2 minutes. Remove the medium, and resuspend the cells completely in 50 µL of water or neutral buffer by pipetting up and down. Add an additional 50 µL of 2× concentrated SDS sample buffer and mix. After induction, repeat the process, adjusting the volume for the increase in optical density. Heat the samples for five minutes immediately before loading 6 – 8 µL onto a denaturing SDS-PAGE gel. We routinely use continuous 15% acrylamide PAGE gels for the analysis of GST N-WASP VCA expression.
6Thawing the yeast cell suspension can be performed more than one way. For any method, it is important to remember to keep the yeast cell suspension cool, to thaw it as evenly as possible and to use it soon after thawing. One suggestion is to place the weighed frozen cell suspension in a 1 L glass beaker with a clean stir bar. Cover the 1 L beaker with aluminum foil and place it in a plastic 4 L beaker with cool water. Hold the 1 L beaker down with a lead ring and place the nested beakers on a stir plate. Once the yeast begins to thaw begin stirring. As the water in the lower beaker is chilled by the thawing cell suspension, replace it with fresh cool (but not cold) water. Alternatively, once the cell suspension is sufficiently thawed, it can be stirred with a serological pipette.
7Lysis of yeast cells requires more aggressive physical methods than does lysis of bacteria. Common ways to lyse yeast cells include grinding in liquid nitrogen, agitation with glass beads, and extrusion at high pressure. The latter can be achieved using the same type of equipment used for bacterial lysis, but efficient lysis requires substantially higher pressures. We lyse yeast cells by passing the cell suspension through a Microfluidics M-110P microfluidizer operated at ~25,000 psi, then repeating for a total of three passes. To address the heat generated, particular care is used to cool the system and lysate. Cooling is achieved by packing much of the system in wet ice, and by stopping the flow each time 90 mL had been homogenized to allow several minutes for the system to cool.
8A common mistake during the ammonium sulfate cut is to add the solid ammonium sulfate too quickly or all at once. This results in locally high concentrations of ammonium sulfate, which can cause undesired proteins to precipitate in a non-equilibrium fashion. A few precautions can minimize this problem.
First, inspect the ammonium sulfate as it is weighed out. Often there are clumps of crystals present, some of which may be larger than 5 mm. If this is a problem, break them up by mashing them with a mortar and pestle. The goal is not to smash the crystals into powder (which does help, but not enough to warrant routinely doing) but just to break up any large clumps of crystals that may be present.
Second, for the amounts described here the addition of ammonium sulfate should occur over 20 to 25 minutes. Time can be scaled down somewhat for smaller volumes. The stirred solution should be checked periodically while the solid is added. Look at the bottom of the beaker, make sure solid ammonium sulfate is not accumulating. If it does, the ammonium sulfate is being added too quickly. Wait a few minutes for the accumulated solid to disperse, then continue to add, but more slowly. A practical way to add the solid is to put half to one third of it in a plastic weigh boat, and to tap it with a spatula. By varying the frequency of tapping a reasonably controllable and uniform addition rate may be found.
9Here, cell suspension weight refers to cells resuspended as in Section 3.1 Step 6, where one third of the suspension weight is wet cell mass. If cells are resuspended at a different cell density, the protocol should be scaled according to the wet cell weight lysed, not according to the cell suspension weight.
10Cut and rinse lengths of 50 kDa cut-off dialysis membrane (Spectra/Por 6, #132544) with ultrapure water. Enough dialysis tubing should be prepared to hold the resuspended volume, plus a 70% increase in volume due to the difference in osmotic strength between the resuspended pellets and buffer A. For the indicated dialysis tubing cut a total of 50 cm per 60 mL, split across at least two pieces. Affix dialysis clips onto one end of the tubing and transfer the resuspended pellets into the dialysis tubing, leaving at least 50% of the length as slack. Close off the other end with additional dialysis tubing clips. Dialyze the pellets against 12 L of buffer A overnight (greater than 6 hours) at 4°C, stirring slowly. It is usually necessary to use two medium or large dialysis clips at either end of the tubing to give the suspension/tubing sufficient buoyancy to prevent it from hitting the stir bar. Alternatively, hang the dialysis tubing from a rod arranged over the dialysis vessel.
11E. coli expressing GST N-WASP VCA can be lysed by a number of common methods. We have used sonication and extrusion using an Avestin EmulsiFlex-C5 cell disruptor. In either case, some care should be used to minimize heating of the sample. When using sonication, use a series of sonication pulses (about 10 seconds total sonication time) and place the solution on ice for 1–2 minutes between pulse series. For extrusion, the cell disruptor has a heat exchanger mounted immediately following the lysis aperture, and chilled water is flowed through this during lysis.
12The DEAE column should be sized according to how much culture is lysed. At least 10 mL of DEAE Sepharose FF should be used per L of LB VCA culture. When lysing volumes larger than 4 L it is usually more practical to repeat the DEAE step and pool the results than to use a larger column.
The DEAE Sepharose column can be reused many times. The principle reason the column should be repacked is accidental introduction of air. Between uses, the column should be thoroughly cleaned. The final high salt wash (in 100% QB1) does a reasonable job removing most protein contaminants, but leaves behind a significant amount of nucleic acid. This can be depleted with washes at different percentages of QB1 (with QA1 making up the balance). Wash the DEAE column with ~1 column volume at 5% QB1, followed by 35%, 65%, 100%, 5%, 35%, 65%, and 5% QB1. If there is still substantial absorbance at 254 nm eluted during the 35% QB1 step, add an additional 1 –2 cycles of 35%, 65% and 5% QB1.
13To save costs, we routinely regenerate Glutathione Sepharose 4B after use in purifying bacterially expressed proteins. The resin is regenerated with 3 column volumes of 6 M guanidine hydrochloride (98% purity), and then washed extensively with water. The resin can be further washed with 2% SDS, although extensive washes with water are necessary between the guanidine and SDS washes. After the initial use, capacity of the resin drops by roughly half, and then is more or less stable for subsequent uses. Regenerated resin can be used here, but adjust the total column volume for the two-fold decrease in capacity. Given the different possible contaminants, once used with yeast lysate glutathione resin is not regenerated but is instead discarded.
14A smaller column such as a 1.5 cm diameter disposable column (e. g., Bio-Rad Econ-Pac #732-1010) is needed when scaling down the prep (~100 g of cells) and less than 2.5 mL of Glutathione Sepharose 4B resin is used. These smaller columns can also be used in the prep described here as an alternative to the 2.5 cm glass Econo-Column. If using for this prep, split the 6 mL of resin into three 1.5 cm columns. In either case, a stopcock and a piece of tubing attached to the bottom of the column will help to keep the solution flowing (due to the increased height between the top of the liquid and the column outlet) and will allow stopping the flow to recover the resin.
15The Saccharomyces cerevisiae Arp2/3 complex ArpC1 subunit (ARC40) has been reported to have an odd mobility on SDS-PAGE gels (30). We have routinely noted this as well. The protein may not appear on the gel at all when samples are prepared in a standard fashion (mixed with 2× reducing SDS loading buffer and heated at 95°C for 3–5 minutes). This seems to be specific to ARC40 at moderate to high concentrations, and is dependent on heating of the SDS-PAGE samples. If it is necessary that ARC40 completely enter the gel, dilute the sample to ~40 nM or less prior to the addition of SDS and reducing agent, and omit the heating of the sample. In many cases, this drops the concentration out of standard coomassie gel staining sensitivity, necessitating silver staining or Western blotting methods.
16Given the length of the second day of the preparation (Section 3.4), once Arp2/3 complex is eluted from the GST-VCA column it is held at 4°C overnight and then exchanged into buffer QC the following day. A desalting column is employed to save time (Section 3.5, steps 1 and 2). If necessary, this allows delaying of SDS-PAGE assessment and pooling until the morning of the third day. Alternatively, dialysis could be set up against 4 L of buffer QC at the end of the second day and steps 1 and 2 of Section 3.5 skipped.
17Our method of quantifying Arp2/3 complex is based on tryptophan UV absorption. An extinction coefficient at 280 nm of 245240 M−1 cm−1 was estimated from the number of tryptophan and tyrosine residues (24 and 76, respectively) in a complex. As the complex may bring an ATP or ADP nucleotide along with it during purification, we routinely measure absorption at 290 nm, and correct for any scatter by using the absorption at 314 nm. By measuring the relative absorbance of a sample judged to be devoid of nucleotide at 280 nm and 290 nm, we found the A290/A280 ratio to be 0.6. Thus, we routinely measure the concentration using an extinction coefficient at 290 nm of 147000 M−1 cm−1. Our experience using laser interference to quantify the complex during analytical ultracentrifugation experiments shows this extinction coefficient to be within 10% of the correct value.
18Directly freezing small aliquots of Arp2/3 complex in liquid nitrogen results in a slight degree of aggregation, and measurable loss of activity. For routine assay at 10 nM of Arp2/3 complex in pyrene actin polymerization assays, we supplement 600 nM Arp2/3 complex in KMEHd-A with one half of a part buffer F (i. e., 5 mL of buffer F is added to 10 mL of 600 nM Arp2/3 complex solution). Small volumes (e.g., 80 µL) can then be placed into small tubes (200 µL thin wall PCR tubes are convenient for this) and snap frozen in liquid nitrogen. For additional commentary, see the bovine Arp2/3 complex purification notes (23).