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

  1. 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.
  2. 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.
  3. 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

  1. 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).
  2. 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.
  3. 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.
  4. 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.
  5. 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 at700g in a swinging bucket centrifuge.
  6. 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.
  7. The resin is washed twice (5 min each atroom temperature, orbit shaker) with 20 ml of freshbuffer, excluding PMSF or-ME.
  8. 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.
  9. 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.
  10. The supernatant is removed and saved (eluted fraction).
  11. 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 7.5-8.0). We measure the absorbtion at 280 and use 28260 (cm-1M-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.

  1. 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.
  2. 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.

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-1M-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 CBD-EGFP 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 “over-labeling” (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 hourat 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 CBD-EGFP (MeroCBD). Fractions of approximately 200 L each are collected. An aliquot (3-5 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 CBD-EGFP 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-1M-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).
  1. 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 environment-sensing, 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.
  1. 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).
  1. 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.
  1. Stop reaction with 5µL β-Mercaptoethanol – mix and incubate at RT for 5 min
  1. 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.
  1. 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 /  max (nm). / 
DMSO / 596 / 135301.00
H2O / 592 / 97929.00
MeOH / 584 / 119760.00
BuOH / 590 / 113963.00

I-BA-s-IA, Molecular Weight 726.62

Solvent /  max (nm) / 
DMSO / 575 / 190910
H2O / 569 / 200564
MeOH / 565 / 168151
BuOH / 569 / 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 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.