Purification of Non-muscle Actin
In the past decade or so, a number of new actin regulatory
proteins and their functions in the motility of
nonmuscle cells have been unveiled. We now understand
in some detail how extracellular stimuli are
transmitted to the microfilament system via
of the phosphatidylinositol cycle, rho-family of
GTPases, causing polymerization of actin through the
recruitment of WASp, WAVE, ARP2/3, or formin proteins.
The polymerization of actin requires constant
supply of actin kept in an unpolymerized form by proteins
such as thymosin, cofilin, and profilin. Filamentcapping,
cross-linking, destabilising, and sequestering
proteins cooperate with monomeric (G)-actin to contribute
to the dynamics of the system. Thus, complex
machineries for the controlled polymerisation of actin
are now being revealed (see Ridley et al.
, 2003; Innocenti et al.
Isoforms of actin differ in their affinities for their
ligands, their functioning, and subcellular locations.
Therefore, to understand the function of actin in nonmuscle
cells, it is important to work with nonmuscle
actin when studying actin-binding proteins (ABPs)
from nonmuscle tissues. Unfortunately, this is often
neglected because of the ready access to α-actin
from rabbit skeletal muscle (Pardee and Spudich,
1982). A further complication is the covalent modification
introduced in α-actin during the commonly
used purification protocol involving acetone extraction
of muscle tissue (Selden et al.
, 2000). This modification
leads to the formation of stable dimers,
explaining fast polymerisation kinetics and other
properties thought to be characteristic for α-actin.
However, i- and -actin are prepared easily from
various nonmuscle tissues using the method described
in this article.
Here, the isolation of β- and γ-actin isoforms from
profilin: actin obtained by affinity chromatography on
-proline) Sepharose is described. It also
describes the purification of yeast and recombinant β-
actin using a yeast expression system and DNase I
affinity chromatography, enabling the production of
large amounts of mutant actins.
II. MATERIALS AND
All chemicals (reagent grade) are from ICN or
Sigma. Hydroxyl apatite is hypatite C of even
number from Clarkson Chemical Co. (Williamsport,
PA). This is the only hydroxyl apatite material we have
used successfully to separate actin isoforms. DNase
I is from ICN (Cat. No. 100579) and poly-(L
is from Sigma (Cat. No. P-2129). CNBr-activated
Sepharose 4B is from Amersham Biosciences (Cat. No.
17-0430). Empty columns are XK-16/20, XK-26/20,
and XK-50/20 from Amersham Biosciences. All other
chromatography equipments and matrices are from
Yeast nitrogen base without amino acids is from
Difco (cat. No. 291940), adenine sulfate is from ICN
(Cat. No. 100195), and the casamino acid vitamin assay
is from Difco (Cat. No. 228820). All other media components
are from Difco. Lysis of yeast cells is achieved
with a purpose-designed bead mill (Innomed Konsult)
using glass beads 0.25-0.5 mm in diameter (Mahlkörper
MK2GX, Willy Bachofen). However, alternative
procedures to break up yeast cells have been described (Jazwinski, 1990). Low-speed centrifugations are
carried out in a Beckman-Coulter Avanti J-20XP with
polypropylene tubes (50 ml; Beckman Cat. No. 357007)
in a Beckman JA-25.0 rotor. Large-volume centrifugations
are performed with polycarbonate centrifuge
bottles (500ml; Beckman no. 335605) in a Beckman
JLA-16.250 rotor. Preparative ultracentrifugations are
done in a Beckman-Coulter L-series centrifuge with
polycarbonate bottles (70ml; Beckman No. 355622) in
Rotor Beckman 45Ti.
A. Preparation of Profilin: Actin Complexes
from Bovine Thymus or Spleen
This protocol is used for the purification of profilin
in complex with a mixture of the cytoplasmic β- and γ-
actin isoforms. The isolation procedure involves affinity
binding to poly-(L
-proline) and chromatography on
hydroxyl apatite to separate actin isoforms, either
alone or in complex with profilin (Segura and Lindberg,
1984; Lindberg, 1988). The yield of profilin: actin
complex is approximately 400 mg/kg of tissue. The following
section describes a preparation starting with
700 g of calf thymus. All buffer volumes and column
sizes refer to that preparation size. If calf thymus
cannot be obtained, calf spleen can be used instead.
Actin isoform distribution is approximately 60% β-
actin and 40% γ-actin in thymus, whereas spleen contains
slightly more β-actin. Spleen is easier to handle,
but contains more proteolytic enzymes, which are
difficult to control.
1. Affinity Purification of Profilin :Actin Complexes
Solutions and Columns
- Homogenisation buffer (2 liters): 10 mM Tris-HCl,
pH 7.6, 0.1M KCl, 0.1M glycine, 1% Triton X-100, and
0.5 mM, dithiothreitol (DTT)
- 2× PLP buffer (3 liters): 20mM Tris-HCl, pH 7.6,
0.2 M KCl, and 0.2 M glycine
- PLP elution buffer (500ml): make immediately
before use. Mix 250ml of 2x PLP buffer with 150ml of
dimethyl sulfoxide (DMSO) and 100ml distilled H2O.
Mixing DMSO with water is an exothermic reaction,
thus the buffer needs to be cooled on ice before being
applied to the column (no DTT).
- HA buffer (5 liters): 5 mM KPO4, pH 7.6
- HB buffer (0.5 liter): 40mM KPO4 pH 7.6, 1.5M glycine, and 0.5 mM DTT
- Saturated ammonium sulfate (4 liters): Add about
1600 g of (NH4)2SO4 to 2 liters of water to make a saturated
solution. Adjust to pH 7.6 with ammonia.
- Poly-L-proline (PLP) affinity matrix: For the preparation
size described here, mix 0.5 g of poly-(L-proline)
to 30 g of CNBr-activated Sepharose 4B for 2 h at room
temperature or overnight at 4°C according to the supplier's
recommendations. Avoid extensive stirring,
as this will have deleterious effects on the matrix.
Monitor the coupling reaction photometrically at
234 nm in supernatants of small samples of the incubation
mixture, removed at intervals. After each use, wash
the matrix immediately with 1 liter of water, followed
by 1 liter of 1% N-lauryl sarcosine, followed with 1 liter
of water. Reequilibrate with PLP buffer containing
0.01% sodium azide. Stored at 4°C, this matrix should
be good for 10-15 preparations and last for 1-2 years.
- Hydroxyl apatite column: Degas 100ml of settled
hypatite-C in 500ml buffer HA. Pack an XK-26/20
column with hydroxyl apatite at a flow rate of
10ml/min. Attach a top adaptor, but allow some
buffer to be present on top of the matrix and equilibrate
the hydroxyl apatite column with 1 liter of buffer
HA at a flow rate of 10ml/min.
: Wear gloves and take extra care when working
with DMSO. Do not use DTT in DMSO-containing
solutions due to the formation of toxic dimethyl
sulfide. All buffers should be freshly degassed when
used. Add DTT only after degassing. All steps are
carried out at 4°C, and all solutions should be at 4°C, unless stated otherwise. Note that Tris buffers are temperature
2. Separation of Profilin-Bound β- and γ-Actin
- Make 5 liters (1×) PLP buffer using the (2×) buffer
stock. Degas all buffers.
- Partially thaw 700 g thymus and remove fat and
connective tissue. Cut the organ into small pieces and
homogenise in a blender using approximately 2
volumes of homogenisation buffer.
- Transfer the tissue extraxt to 500-ml polycarbonate
bottles and centrifuge at 20,000g (12,000rpm with
the JLA-16.250 rotor) for 30min.
- Filter the supernatant through glass wool,
transfer to 70-ml polycarbonate bottles, and centrifuge
at 100,000g (30,000rpm with the 45TI rotor) for
- Prepare the PLP column in a laminar flow hood:
Pack an XK-50/20 column with the affinity matrix
described in step 6 of the previous section and equilibrate
it with PLP buffer at a flow rate of 5-10ml/min.
- Filter the supernatant from step 4 through glass
wool and load it onto the PLP column.
- After all the protein has been loaded, wash the
matrix with PLP buffer until the color is removed from
- Elute bound proteins with ice-cold PLP elution 0.4
buffer into a 500-ml measuring cylinder on ice. After
this step the preparation already contains mainly profilin
and profilin:actin complexes.
- Dilute the PLP eluate with 2 volumes of buffer
HA and load the solution onto the hydroxyl apatite
column at a rate of 10ml/min, monitoring the UV
absorbance at 280nm. The DMSO accounts for a small
increase in absorbance at 280nm. A tough layer may
form on top of the matrix bed, restricting buffer flow.
If necessary, stop the pump, disconnect the top
adaptor, and resuspend the top centimeter of matrix
bed by careful stirring with a glass pipette. Allow the
matrix to resettle, replace the top adaptor, and continue
- After the protein has entered the column, wash
with buffer HA to remove DMSO. Finish washing
when a low baseline is reestablished. Carefully press
the top adaptor onto the gel matrix.
- For separation of profilin: β-actin from profilin:
γ-actin complexes, elute the hydroxyl apatite-bound
material with a linear gradient of 5mM potassium
phosphate to 40 mM phosphate/1.5 M glycine. Employ
a gradient mixer containing 350ml HA in the start
buffer compartment and 350ml HB in the finishing
buffer compartment. (If a different column dimension
is used, the gradient length should be approximately
12 column volumes.) Elute the protein at a flow rate of
0.3 ml/min. Collect 10-min fractions.
- Identify the fractions containing profilin: γ-actin
and profilin: β-actin and pool the appropriate fractions
(see Fig. 1). Continue as described in step 14.
|FIGURE 1 Separation of profilin, profilin : β-actin, and profilin :
γ-actin using hydroxyl apatite chromatography
(see Section III,A).
The slightly more basic γ-actin : profilin complex elutes at lower
ionic strength, followed by the
β-actin : profilin complex at higher
phosphate/glycine concentration. The small initial peak contains
3. Elution of Total PA
4. Ammonium Sulfate Precipitation of PA
- If isoform separation is not aspired, elute the
hydroxyl apatite-adsorbed protein using HB buffer.
The eluate consists of mixed actin isoforms in complex
with profilin, plus a slight excess of profilin.
- Precipitate the protein by dialysis against 2
liters of saturated ammonium sulfate using dialysis
tubing with a molecular mass cutoff under 12 kDa. The
resulting protein is suitably stored as a precipitate in
ammonium sulfate. Stored in this form at 4°C the PA
is stable for 6-12 months. Estimate the concentration
of protein by diluting 25-50µl of the well-suspended
solution into 1 ml of water and determine the
absorbance at 280 and 310nm (280-310 roughly corresponds
to milligram per milliliter).
The dialysis against saturated ammonium sulfate
results in a reduction of the sample volume, ensuring
effective protein precipitation. It is important to use
enough saturated ammonium sulfate to avoid dilution
of the salt below 40-50% saturation, which is the concentration
precipitating the profilin: actin.
For efficient extraction of the tissue, it may be
helpful to pass the organ through a meat grinder
before homogenisation in a blendor. The buffer over
tissue ratio should not be lower than 2:1. Continue
homogenisation until no chunks are visible, which
should take about 2 to 3min. Do not continue
homogenisation over prolonged periods of time.
Avoid excessive foaming.
5. Isolation of Profilin and Actin from Precipitated
P :A Complex
Solutions and Columns
6. Purification of Actin
- G buffer (2 liters): 5 mM Tris-HCl, pH 7.6, 0.5 mM ATP, 0.1 mM CaCl2, and 0.5 mM DTT
- Resuspension buffer (0.1 liter): G buffer, add DTT
- F buffer (0.1 liter): 5 mM Tris-HCl, pH 7.6, 0.5 mM ATP, 2 mM MgCl2, 50 mM KCl, and 0.5 mM DTT
- 2 M KP04 pH 7.6 (make 50ml)
- P buffer (1 liter): 10 mM KPO4 pH 7.6, and 0.5 mM DTT
- Prepare an XK-26/70 Sephacryl-300 gel filtration
column equilibrated with G buffer
- Prepare an XK-26/50 Sephadex G-25 gel filtration
column equilibrated with P buffer
- Prepare an XK-16/10 DEAE-Sephadex anionexchange
column equilibrated with P buffer
7. Purification of Profilin
- Collect 30-50mg of ammonium sulfateprecipitated
profilin:actin (either isoform; previous
section, step 14) by centrifugation in a JLA-16.250 rotor
at 10,000rpm for 10 min. Discard the supernatant,
leave the tubes upside down for a few minutes, and
then wipe the inside of the tubes with Kleenex to
remove residual ammonium sulfate.
- Dissolve pellets in a small volume of resuspension
buffer, stir, and add buffer until the solution
becomes clear. Protein concentration should be 10-
20mg/ml. Centrifuge the solution again (as in step 1)
to remove undissolved residues. Save the supernatant.
- Measure the volume of the protein solution and
add MgCl2 to 5 mM and EGTA to 0.5 mM. Allow actin
to polymerise at room temperature for 30min.
- Induce paracrystal formation by the addition of
1 volume of 2M KPO4. Mix carefully; avoid extensive
pipetting. Leave for 1 h at room temperature or
overnight at 4°C.
- Collect the actin paracrystals by centrifugation at
10,000g for 20min at 15°C. The supernatant contains
profilin and small amounts of remaining profilin:
actin; save supernatant on ice for the profilin purification
described later. The pellet contains actin
- Resuspend the paracrystals in F buffer to dissolve
the actin filaments. Mix by careful pipetting until
all lumps have dissolved.
- Sediment filaments by ultracentrifugation at
100,000g for 3 h at 15°C. Remove the supernatant and
add a small amount of G buffer to the F-actin pellet
and leave it for 10-20min. Seal the tip of a Pasteur
pipette and use it to scrape the pellet off the walls of
the tube. Carefully transfer the pieces of F-actin gel to
a Dounce-type glass homogeniser and homogenise
without introducing air. Measure the protein concentration
using an extinction coefficient at A290-310 of
0.63 ml/mg × cm. Dilute the F-actin to 5 mg/ml to get
- Dialyze the actin sample against 10 volumes of G
buffer in 12- to 14-kDa cutoff tubing with three buffer
changes and homogenisations. Four to 6 h of dialysis
is sufficient for every buffer change.
- Subject the dialyzed actin to ultracentrifugation
at 100,000g for 3 h at 4°C in order to remove undissolved
material. Load the supernatant onto the
Sephacryl S-300 column. The sample volume should
not exceed 4% of the column volume. Elute with G buffer at a flow rate of 30ml/h. Collect 4- to 6-ml fractions.
Measure protein concentration in the fractions
and pool the protein peak. Analyze by SDS-PAGE,
drop freeze (20 µl droplets) the actin in liquid N2, and
store in a nitrogen tank.
- Desalt the profilin solution from step 5 by gel
filtration over Sephadex G-25. The volume of the
protein solution may be up to approximately 30% of
the column bed volume. Collect 2- to 3-ml fractions.
- Determine protein concentrations across the
chromatogram. Measure conductivity in the last fractions
to make sure the solution will not be contaminated
by salts and pool the protein-containing
12. Run the G-25 pool over the small DEAESephadex
column. Contaminating actin will stay on
the ion exchanger. The column flow through contains
pure profilin. Quick-freeze aliquots in liquid N2 and
store at -80°C.
Before inducing paracrystal formation (step 4),
check on the progress of the polymerisation reaction
by rocking the tube carefully; the protein solution
should have a gel-like consistence. If necessary, stimulate
polymer formation by a few short bursts in an
Polymers of cytoplasmic actin are more cold sensitive
than polymers of α-actin. Therefore, the ultracentrifugation
step that aims at collecting actin filaments
is carried out at 15°C.
B. Purification of Recombinant β-Actin from
This protocol is used for the large-scale preparation
of recombinant actin expressed in Saccharomyces cerevisiae
strain K923 using the temperature-inducible
expression system described by Karlsson (1988). The
heterologous actin cDNA is placed under the combined
control of the PGK promoter and the negative
transcription factor α2 and is introduced into strain
K923 containing a temperature-sensitive mutation in
the mating type switch (Walton and Yarranton, 1989;
see also Sledziewski et al.
, 1988). This results in temperature-
inducible expression of the heterologous
actin. At 34°C, the cells are MATα and produce the α2
protein, which blocks transcription of the cloned
cDNA. By lowering the temperature to 23°C, the cells
switch mating type and cease to produce α2, leading
to expression of the recombinant actin. Total actin is isolated from yeast extracts by DNase I affinity chromatography
(Zechel, 1980), and the recombinant
isoform is separated from the endogenous actin by
hydroxyl apatite chromatography (Karlsson, 1988).
Ten liters of flask culture produces 120-150g of yeast
cells (the amount needed for a preparation as described
later), yielding in the range of 10-15mg each
of yeast and recombinant actin. Usually, a fermentor
culture in 10-liter fermentors is used, producing
700-800 g of yeast, which is material for five to six standard
preparations of recombinant actin. The fermentation
process has successfully been scaled to 50-100
liters. It should be noted that the yeast does not modify
the N terminus of actin and does not methylate residue
His73 (for discussion, see Nyman et al.
This method has been adapted for the production of
human profilin (Aspenström et al.
, 1991) and should
also work for other proteins.
1. Fermentor Culture of Yeast Expressing
- S. cerevisiae strain K923: HMLα, mat::LEU2+ hmr ::TRP1+ ura3, ade2, sir3ts; MATa at 23°C, MATα at
34°C. This strain is grown on standard YPD medium
(Sambrook and Russell, 2001) at 34°C.
- UYM media for transformed yeast strain K923:
Bottle 1:8 g yeast nitrogen base without amino acids,
55 mg adenine sulfate, 55 mg L-Tyr; add H2O to 580 ml.
Bottle 2:11 g casamino acid vitamin assay; add H2O to
300ml. Autoclave, allow to cool. Combine contents of
bottles 1 and 2 and add 100ml 20% glucose, 10ml 0.5%
L-Trp, and 10ml 1% L-Leu. Grow the transformed
strain on min-ura plates at 34°C.
2. Purification of Recombinant Actin from Yeast
Solutions and Columns
- Culture K923 transformed with pY-β-actin to
single colonies on a min-ura plate at 34°C. Inoculate
into 10ml UYM and culture for 24h under vigorous
shaking at 34°C, transfer into 1 liter of UYM, and continue
culturing for another 24 h at 34°C.
- Transfer the inoculum into the fermenter, which
has been prepared with 9 liters modified YPD [220g
peptone, 220 g yeast extract, 2.2 g adenine sulfate, 2.5 g
glucose (0.25%), and PPG to avoid foaming]. Glucose
is added continuously from a 50% stock, adjusting the
concentration with the density of the culture (measured
by OD640) to keep it at approximately 0.5%.
Approximately 12h after inocculation, add 220g of
yeast extract in 500ml water. Keep the culture at 34°C until OD640 is between 1 and 2 (this takes 6-8 h). At this
stage, lower the temperature to 23°C to initiate the
mating-type switch, which induces production of the recombinant protein. If larger scale (50-100 liters) fermenations
are required, prepare the inoculum in the
fermentor at a volume of 5-10 liters in UYM medium
before transferring into a bigger fermentor with medium
as described earlier.
- Harvest the culture when OD640 no longer increases
(ca. 24-26 h after inocculation). Rapidly cool the
culture to below 10°C, start collecting it into ice-cold
l-liter centrifuge flasks, and centrifuge at 5000rpm for
20 min (Sorvall RC-3 or equivalent). Collect all material
into one or two flasks, centrifuge at 5000 rpm for 30 min,
aliquot into suitable portions (150 g) for preparation of
the protein, and store at -70°C.
- 2× G buffer (3 liters): 10mM Tris-HCl, pH 7.6,
0.2 mM CaCl2, and 1 mM ATP
- G buffer: use 2.5 liters of the 2xG buffer to make
5 liters (5 mM Tris-HCl, pH 7.6, at 4°C, 0.1 mM CaCl2,
and 0.5 mM ATP)
- Na-acetate buffer (0.5 liter): 0.5M sodium acetate,
0.5 mM ATP, pH 7.6, 0.1 mM CaCl2, 10% glycerol, and
0.5 mM DTT
- DNase wash buffer Elu-I: use the 2×G buffer to
make 0.5 liter Elu-I: 5 mM Tris-HCl, pH 7.6, at 4°C, 0.1 mM CaCl2, 0.5 mM ATP, 10% formamide, 10% glycerol,
and 0.5 mM DTT
- DNase elution buffer Elu-II: Use the 2×G buffer to
make 0.3 liter Elu-II: 5 mM Tris-HCl, pH 7.6, at 4°C, 0.1 mM CaCl2, 0.5 mM ATP, 40% formamide, 10% glycerol,
and 0.5 mM DTT
- HA buffer (0.5 liter): 5mM KPO4, pH 7.6, and
0.5 mM DTT
- HB buffer (100 ml): 40 mM KPO4, pH 7.6, 1.5M glycine, and 0.5 mM DTT
- Water-diluted protease inhibitor mix (1000 × stock):
0.5mg/ml each of antipain, leupeptin, pepstatin A,
chymostatin, and aprotinin. Store 0.5-ml aliquots at
- Ethanol-diluted protease inhibitor mix (100 × stock):
0.1M phenylmethyl-sulfonyl fluoride (PMSF) 1 mM benzamidine-HCl, and 0.1mg/ml phenanthroline.
Store 5-ml aliquots at -20°C.
- RNase A (500 × stock): 10mg/ml in 0.1M sodium
acetate, pH 5.0. Boil in a water bath for 20min. Store
1-ml aliquots at -20°C.
- ATP for Hypa fractions: 10mM ATP in 40mM K2HPO4; gives a pH of approximately 7.0
- D buffer (1 liter): 10mM Tris-HCl, pH 7.6,
150mM NaCl, 0.5 mM CaCl2, and 0.01% sodium azide
- DNase I affinity matrix: Couple 0.3 g of DNase I
to 15g of CNBr-activated Sepharose 4B overnight at 4°C according to the supplier's recommendations.
Include 1 mM CaCl2 in all buffers. Monitor the coupling
reaction photometrically at 280 nm (see step 6 of
Section III,A). According to our experience, this matrix
will have a capacity of approximately 40mg actin and
will allow purification of actin from up to 150g (wet
weight) of yeast coexpressing recombinant actin. Store
the matrix in D buffer containing 0.01% sodium azide.
If column capacity decreases after several preparations,
wash off bound contaminants with 4M guanidine
hydrochloride, 0.5M sodium acetate, and 30%
glycerol. Kept at 4°C, this matrix should be good for
10-15 preparations and last for 1-2 years. Also note
that long-term exposure of DNase I to reducing agents
inactivates the enzyme.
- Hydroxyl apatite column: Degas 20ml of settled
hypatite C. Pack an XK-16/20 column with the matrix
and equilibrate with HA buffer at 120ml/h.
- Use up to 150g (wet weight) of yeast cells (corresponding
to 10-12 liters of flask culture). Make
500ml lysis buffer by adding water-diluted and
ethanol-diluted protease inhibitors and 50 gl
polypropylene glycol. Thaw the pelleted yeast in
100ml lysis buffer. Lyse the yeast cells by passage
through the bead mill and wash out the lysate using
the remaining lysis buffer.
- Add RNase A. Centrifuge the collected yeast
lysate in a large-volume rotor at 12,000rpm for 40min
at 4°C. Carefully decant the supernatant and filter it
through glass wool.
- Pour an XK-50/20 column using the DNase affinity
matrix (step 13 of the previous section). Equilibrate
the DNase column with G buffer. Pump the yeast
lysate onto the DNase column. Wash with G buffer
until a stable baseline is established as judged by
OD290. Wash consecutively with 5 column volumes of
Na-acetate buffer, wash buffer Elu-I, and G buffer.
- Prepare 200ml of dilution buffer (G buffer containing
10% glycerol). Arrange a measuring cylinder
with 100 ml dilution buffer on a magnetic stirrer. Blank
a spectrophotometer with buffer Elu-II. Reduce the
pump flow rate and start elution with buffer Elu-II.
Follow the A290; at 0.05-0.1, start collecting into the
dilution buffer. The volume of dilution buffer should
be at least 50%. Add more dilution buffer if necessary.
Stop collecting when the A290 of the eluate is below 0.1.
- Apply the diluted actin eluate onto the hydroxyl
apatite column at a flow rate of max 100ml/h, i.e.,
somewhat slower than the packing flow rate, to avoid
further packing of the matrix with the more viscous
- Prepare a fraction collector with 3-ml tubes containing
100-gl aliquots of buffered 10mM ATP (for a
final ATP concentration of 0.5 mM). Start collecting 2-
ml fractions. Elute the actin isoforms using a gradient
of 60ml each of buffers HA and HB at a flow rate of
- Identify actin-containing fractions by measuring
the absorbance at 290nm (Fig. 2), DNase I inhibition
activity, SDS-PAGE, or dot blotting. Pool the fractions
and dialyse against G buffer; alternatively, the pools
may be concentrated and subjected to gel filtration.
|FIGURE 2 Separation of yeast actin and recombinant nonmuscle
β-actin using hydroxyl apatite chromatography (Section III,B). The
more acidic β-actin elutes at the higher phosphate/glycine concentration.
Solid line represents the conductivity.
The protease inhibitor PMSF is counteracted by
reducing agents. Therefore, DTT should be excluded
from the lysis buffer and added to buffers only after
the yeast extract has passed over the DNase column.
Failure to wash the DNase affinity column with
high ionic strength (sodium acetate) buffer will result
in contamination by a number of actin-binding proteins,
with the most prominent one being yeast cofilin.
Prolonged exposure to 20% formamide will lead to
actin denaturation. Therefore, it is important to collect
the actin eluate into at least an equal volume of dilution
buffer and apply it to the next column as quickly
as possible. Attempts to store quick-frozen actin in this
buffer have been unsuccessful.
The most probable reason for poor isoform separation
is too high a flow rate during elution.
The aforementioned conditions for the separation of
recombinant β-actin from yeast actin are not necessarily
applicable to mutants of β-actin or other recombinant
actins (e.g., Aspenstr6m and Karlsson, 1991).
Therefore, it may be necessary to alter conditions.
Aspenström, P., and Karlsson, R. (1991). Interference with myosin
subfragment-1 binding by site-directed mutagenesis of actin. Eur.
Aspenstr6m, P., Lassing, I., and Karlsson, R. (1991). Production, isolation
and characterization of human profilin from Saccharomyces
cerevisiae. J. Muscle Res. Cell Motil
De La Cruz, E. M., Mandinova, A., Steinmetz, M. O., Stoffier, D.,
Aebi, U., and Pollard, T. D. (2000). Polymerization and structure
of nucleotide-free actin filaments. J. Mol. Biol
Gorbunoff, M. J. (1990). Protein chromatography on hydroxyapatite
columns. Methods Enzymol
Innocenti, M., Zucconi, A., Disanza, A., Frittoli, E., Areces, L. B.,
Steffen, A., Stradal, T. E., Di Fiore, P. P., Carlier, M. R, and Scita,
G. (2004). Abil is essential for the formation and activation of a
WAVE2 signalling complex. Nature Cell Biol
Jazwinski, S. M. (1990). Preparation of extracts from yeast. Methods
Karlsson, R. (1988). Expression of chicken beta-actin in Saccharomyces
cerevisiae. Gene 68
Lindberg, U., Schutt, C. E., Hellsten, E., Tj/ider, A. C., and Hult, T.
(1988). The use of poly(L-proline)-Sepharose in the isolation of
profilin and profilactin complexes. BBA 967
Nyman, T., Schiller, H., Korenbaum, E., Schutt, C. E., Karlsson, R.
and Lindberg, U. (2002). The role of MeH73 in actin polymerization
and ATP hydrolysis. J. Mol. Biol
Pardee, J. D., and Spudich, J. A. (1982). Purification of muscle actin. Methods Enzymol 85
Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg,
M. H., Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003). Cell
migration: Integrating signals from front to back. Science 302
Sambrook, J., and Russell, D. (2001). "Molecular Cloning: A
Laboratory Manual." 3rd Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Segura, M., and Lindberg, U. (1984). Separation of non-muscle
isoactins in the free form or as profilactin complexes. JBC 259
Selden, L. A., Kinosian, H. J., Estes, J. E., and Gershman, L. C. (2000).
Cross-linked dimers with nucleating activity in actin prepared
from muscle acetone powder. Biochemistry 39
Sledziewski, A. Z., Bell, A., Kelsay, K., and MacKay, V. L. (1988). Construction
of temperature-regulated yeast promoters using the
MATα2 repression system. Bio/Technology 6
Walton, E. E, and Yarranton, G. T. (1989). Negative regulation of
gene expression by mating-type. In
"The Molecular and Cell
Biology of Yeasts" (E. E Walton and G. T. Yarranton eds.), pp.
43-69. Blackie, Glasgow, UK.
Zechel, K. (1980). Isolation of polymerization-competent cytoplasmic
actin by affinity chromatography on immobilized DNase I
using formamide as eluant. Eur. J. Biochem. 110