Additional notes re CBD (Cdc42 activation biosensor)
This protocol refers to the biosensor described in the following paper: Nalbant, P.,
Hodgson, L., Kraynov, V., Toutchkine, A., and Hahn, K.M. Activation of
Endogenous Cdc42 Visualized in Living Cells. Science, 305:1615-1619, 2004.
The following procedures are very detailed, but are actually quite straightforward.
This biosensor is made by expressing a protein and then covalently attaching a
dye. We have published synthesis of one such dye (Toutchkine, A., Kraynov, V.,
and Hahn, K.M. Solvent-Sensitive Dyes to Report Protein Conformational
Changes in Living Cells, J. Amer. Chem. Soc., 125:4132-4145, 2003.). Please
contact Klaus Hahn if you intend to make the biosensor, as we are improving the
dyes and will likely have more recent improved versions.
Contents:
1. protein expression and purification
2. labeling with I- SO IAA dye
3. generic dye labeling protocol
4. note re photobleach correction
1. Protein Expression and Purification
CBD-EGFP is expressed in the form of C-terminal 6xHis fusion from the
prokaryotic expression vector pET23. This vector has a strong T7 promoter, and is
designed to work with BL21(DE3) strains of E.coli (Stratagene). It was determined
experimentally that the highest levels of expression are observed when a plain T7
promoter (not T7lac) is used in combination with a BL21(DE3) strain and not
BL21(DE3)pLysS, which allows for leaky expression. The protein is induced and
expressed at room temperature (26C), to increase the portion of the correctly folded,
soluble CBD-EGFP.
Key points include:
1) The bacteria strain is critical. We use BL21(DE3). Do not use BL21(DE3)pLysS.
2) Use Talon resin (Co2+ affinity, Clontech Inc.). Do not use Ni-NTA resin. Use
approximately 2 ml Talon resin (dry volume) for 3g of cell pellet.
3) Use the suggested buffers in this article.
4) Use enough buffer during lysis; i.e., for 3g cell pellet use 35 ml total lysis buffer.
Day 1
1. Competent BL21(DE3) cells are transformed with pET23-CBD-EGFP according
to standard protocols {Sambrook, 1989 #5531}, and plated on LB-carb (100
g/ml carbenicillin) plates. We usually split the 200l transformation volume
over 2 plates.
2. Plates are incubated at 37C overnight.
Day 2
3. 500 ml of LB-carb (100 g/ml carbenicillin) are inoculated with the colonies from
the plates. 5 ml of media are added on each plate and cells are resuspended into
the media. The cell suspension is transferred into the 500 ml LB-carb and grown
in a shaker at 37C, 225rpm to OD600 = 0.8-0.9. The culture is briefly chilled on
ice to 26C and put back in the shaking incubator turned down to 26C.
4. IPTG (1 M stock in water, kept at –20C) is added to a final concentration of 0.2
mM, and the cultures are allowed to grow for another 6 hours at 26C at 225rpm.
IPTG concentrations of 0.2~0.5mM has been used successfully.
5. Cells are collected by centrifugation (Beckman J-6M rotor, 20 min, 4,000 rpm),
and stored as a pellet at –20C until use. Approximately 2.5-3 g of cells is usually
obtained from each liter of culture.
Day 3
6. Cells (~3 g) are resuspended in 35 ml of the Lysis buffer [50 mM NaH2PO4, pH
7.6, 300 mM NaCl, 10% glycerol, 5 mM MgCl2, 2 mM -ME, 1 mM PMSF] and
lysed by sonication (4 pulses, 30 sec each on ice with 1 min rests).
7. The lysates are centrifuged at 13,000 rpm for 30 min, and the supernatant
containing CBD-EGFP is carefully transferred into a 50 ml Falcon tube.
8. While the lysates are being centrifuged, 2 ml of Talon resin (dry volume) is
transferred into a 50 ml Falcon tube and centrifuged at 700g in a swinging bucket
centrifuge. We use 2 ml resin per 3 g cell pellet.
9. Talon resin is washed twice with 10 volumes of the lysis buffer (no -ME and
PMSF) in a 50 ml Falcon tube. Again, pellet the resin by centrifuging at 700g.
10. The cell lysate is added to the washed Talon resin in the 50 ml falcon tube and
inverted gently using an orbit shaker at room temperature for 40 min ~ 1hr,
wrapped in foil to avoid unnecessary exposure of EGFP to light. The resin is then
separated by centrifugation at 700g in a swinging bucket centrifuge.
11. The supernatant is removed and saved (unbound fraction). If a large portion of
unbound material is present in this fraction, consider increasing the Talon resin
volume by preparing 2 tubes of 2ml resin and splitting the lysates into 2 tubes
during the binding reaction.
12. The resin is washed twice (5 min each at room temperature, orbit shaker) with 20
ml of fresh buffer, excluding PMSF or -ME.
13. Final wash is performed with 10 volumes of buffer containing 5 mM imidazole. It
is convenient to prepare freshly, 5ml of stock 1M imidazole solution in the same
buffer. Always prepare the stock imidazole solution fresh.
14. The elution is performed by adding 5 ml buffer containing 150 mM imidazole to
the resin and rotated using the orbit shaker at room temperature for 5 min. Pellet
the resin again by centrifugation.
15. The supernatant is removed and saved (eluted fraction).
16. The resulting 5 ml eluate is concentrated with the Ultrafree – 4 Centrifugal
Filteration Device (Millipore; 5000Da cut-off) by centrifugation at 4 C following
the manufacturer’s directions. Check the concentration process every 20 min to
ensure proper filtration. Do not over-concentrate; optimal final concentration
should be approximately 120 M. The concentration of CBD-EGFP is measured
by taking a small aliquot (5-10 L) and diluting into 50 mM Tris HCl (pH 7.58.0). We measure the absorbtion at 280 and use 28260 (cm-1 M-1) as extinction
coefficient for the protein, using the following equation:
[CBD-EGFP] (in mol/L) = (OD280*dilution factor) / 28260
(1)
On average, 10-15 mg of CBD-EGFP is obtained per liter culture.
17. If dye-labeling is to be performed the following day, a part of the concentrated
protein is dialyzed overnight against 2 L of 50 mM NaH2PO4 (monobasic sodium
phosphate) buffer at pH 7.5. Slide-A-Lyzer cassette (PIERCE) with a molecular
weight cut-off of 3,500Da is used.
18. For long term storage, the protein is dialyzed over night against 2 L of storage
buffer [50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 10% Glycerol] and
is flash frozen on dry ice or liquid nitrogen.
19.
2. Labeling with I-SO IAA dye
The following protocol from our lab describes labeling of the CBD Cdc42 sensor. A
more generic protocol for biosensor labeling is also included following this.
For attachment of the dye to cysteine, CBD-EGFP needs to be in 50 mM
NaH2PO4 buffer, pH 7.5 at a protein concentration of 100 M. A fresh solution of dye is
prepared in pure DMSO by adding approximately 1 mg of dye into 30-40 L DMSO.
Once dissolved in DMSO, dye cannot be kept more than 12hrs. The exact concentration
of the dye is determined spectrophotometrically by diluting the DMSO solution 1:5000 in
methanol. The ISO-IAA dye extinction coefficient in methanol at maximum absorption
(610 nm) is 125,000 (cm-1 M-1).
[ISO-IAA] (in mol/L)= (OD610*dilution factor) / 125000
(2)
We routinely obtain concentrations of 20~25 mM for the DMSO stock solution.
A 300 L aliquot of fresh CBD-EGFP protein is transferred into a 2 ml Eppendorf
tube wrapped in foil to protect from light. The dye is added in 2-3 aliquots to the CBDEGFP solution to make the final dye to protein ratio in the reaction to 5:1 or 6:1. The
protein-dye mix is vortexed once briefly at highest setting immediately after addition of
the dye stock solution. Using higher dye to CBD-EGFP ratios (e. g. 10:1 or 15:1) in the
reaction mixture can produce excessive amounts of precipitated material and “overlabeling” (dye to protein ratio in the purified covalent adduct greater than 1.0). The
optimal dye to protein ratio depends on the reactivity of the dye, so it can vary with
different dyes and/or dye preparations. The tube containing the reaction mixture is
wrapped in foil and gently rotated on a rotating wheel for exactly 1 hour at room
temperature. Here, the time is critical since the dye is very reactive; therefore longer
reaction times result in over-labeling. After the reaction is complete, 1~5 l -ME is
added to the stop the reaction and the tube is incubated for 5-10 min at room temperature
on the rotating wheel. The tube is centrifuged at room temperature to pellet insoluble
material (i.e, 2 min at 13,000 rpm in an Eppendorf benchtop microcentrifuge). The
supernatant is then loaded on a small G25 gel filtration column (0.5 cm x 6-8 cm) to
separate the conjugate from free dye. This column is equilibrated and run in 50 mM
NaH2PO4 buffer at pH 7.5. The first colored band to elute contains the dye-labeled CBDEGFP (MeroCBD). Fractions of approximately 200 L each are collected. An aliquot (35 L) of each fraction is analyzed by 12% SDS-PAGE to confirm the presence and purity
of the MeroCBD. Fluorescence visualization of the gel assures labeling with the dye.
Protein concentration can be determined by taking an absorbance spectrum of the
conjugate solution. Dilute a small aliquot (5-10 L) in 50mM Tris HCl, pH 7.5-8.0 and
use OD280 for the calculation using the Equation (1). However, the protein absorbance at
280 overlaps some portions of the dye absorbtion. One can determine the protein
concentration independently by comparing a sample of MeroCBD to unlabeled CBDEGFP using different sample concentrations. Colorimetric assays, limited to those with
readouts that do not overlap dye absorbance, have been more variable in our hands.
The dye concentration is determined by taking an absorbance spectrum of
MeroCBD. 5~10l of MeroCBD is diluted in DMSO. DMSO as a solvent will
overwhelm effects of the protein on the dye absorbance to result in a consistent dye
extinction coefficient. OD at the dye absorbance maximum of 610nm is used to calculate
the dye concentration using the Equation (2) with the extinction coefficient of I-SO-IAA
in DMSO (145,000 cm-1 M-1).
We routinely recover approximately 40-60% of the CBD-EGFP as purified
MeroCBD, due to some precipitation during labeling and loss during gel filtration. The
final eluate concentration is usually 50-60 M. Labeling efficiency under these
conditions varies between 0.7-0.9 dye to protein ratio. MeroCBD solution is aliquoted
into 15-20 l aliquots and flash frozen with dry ice or liquid nitrogen and stored at –80C.
Alternatively, the MeroCBD may be kept at 4C for up to one week.
3. “Generic” protein labeling protocol
1.
Dissolve a small amount dye in 50µL DMSO (a few mg. Do not try to weigh it –
static charge will likely cause you to lose much of the dye).
2.
Dilute in DMSO until the absorbance max at the long wavelength dye peak ( above
450 nm, at 500-700 nm for most of our dyes) is below 0.2, for accurate
measurement. Determine the concentration of the dye in this solution using the
dye extinction coefficient in DMSO. Note that these dyes are environmentsensing, so the extinction coefficient is solvent dependent. Do not do this
procedure in some other solvent and use the DMSO extinction coefficient. This
step is meant to determine the concentration of the solution produced in step 1. It
is that which will be added to the protein, not the dilute solution produced in this
step to determine the dye concentration.
3.
In the protein reaction mixture, the protein/dye ratio should be about 1:5 (for IAA).
Add the original concentrated solution of dye to the protein dropwise, with gentle
mixing between drops. We try to minize the percent DMSO in the protein solution.
That is why we use a concentrated dye solution. The concentration of the protein
is important – too dilute a protein will slow reaction. We typically use a 300µL
reaction volume (buffer: 50mM sodium phosphate, pH 7.4) with 100µM protein
(~5mg/ml).
4.
Incubate (with mixing in a shaker or rotating platform) @ room temperature for 2 h.
Note that reaction conditions (time, temperature, pH, concentrations, dye:protein
ratios) can be varied to accommodate less stable proteins or to affect the number
and site of labeling. For iodoacetamide or bromoacetamide dyes, use pH 6.5-7.5.
5.
Stop reaction with 5µL β-Mercaptoethanol – mix and incubate at RT for 5 min
6.
Separate unreacted dye using G-15 or G-25 gel filtration columns as appropriate.
Use long thin columns rather than short fat ones, Use column volume OF 20X
over loading volume.
7.
To determine labeling efficiency, the labeled protein is diluted in DMSO and the
absorbance is measured at the absorbance maximum of the dye (to determine dye
concentration) and protein concentration is determined by SDS-PAGE (through
comparison with serial dilutions of known standards), by a colorimetric assay that
does not overlap the dye, or other means. Be aware that in colorimetric assays the
dye will affect absorbance measurements at 200-300 nm, in addition to the long
wavelength absorbance peak of the dye.
8. Notes: Dyes should be used immediately after preparing stock solutions, or at most
within a few hours. Do not store the dyes as solutions – even in frozen solutions at 80 degradation occurs (we are trying to understand this).
I-SO-s-IA, Molecular Weight 724.63
Solvent
DMSO
H2O
MeOH
BuOH
 max (nm).
596
592
584
590

135301.00
97929.00
119760.00
113963.00
I-BA-s-IA, Molecular Weight 726.62
Solvent
DMSO
H2O
MeOH
BuOH
 max (nm)
575
569
565
569

190910
200564
168151
193519
4. Notes re photobleach correction
Hahn lab has published a Methods in Enzymology article describing photobleach
correction for this Cdc42 biosensor . The Matlab routines it references can be found at the
Hahn lab web page, accessed via http://www.med.unc.edu/pharm/. The article is:
Hodgson, L. Nalbant, P. Shen, F., and Hahn, K. Imaging and photobleach correction of
Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol., 406:140-156,
2006.