Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

Protocolo

NOTE: An overview of this protocol is shown in Figure 1.

1. Expression and purification of GST-FKBP51 and GST-FKBP52 (Figure 1A)

1. Plasmids
   NOTE: Obtain cDNA clones for human FKBP51 (clone id: 5723416) and for human FKBP52 (clone id: 7474554) from IMAGE consortium.
   1. Amplify the human FKBP51 DNA by PCR with primers (forward; 5`GGATCCATGACTACTGATGAAGGT-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing BamHI and XhoI overhangs and clone into pGEX6-1 vector at BamHI / XhoI restriction sites.
   2. Amplify the human FKBP52 DNA by PCR with primers (forward; 5`-GAATTCATGACAGCCGAGGAGATG-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing EcoRI and XhoI overhangs and clone into pGEX6-2 vector at EcoRI / XhoI restriction sites.
      NOTE: PCR reaction set up and conditions are shown in Table 1 and Table 2.
   3. Verify the inserted sequence and transform the plasmids into the chemically competent E. coli according to the manufacture protocol.
2. Protein expression and purification
   1. Add 25 g of Luria broth (LB) base in 1 L of distilled water to make the LB solution. Autoclave it at 121 °C for 15 min. After cooling, add 50 µg/mL ampicillin.
   2. Take a colony of bacteria expressing GST-FKBP51 or GST-FKBP52 and mix with 500 µL of LB solution in a 1.5 mL tube. Vortex.
   3. Add the mixture of "1.2.2" into 1 L of LB solution in the Erlenmeyer flask covered with an aluminum foil. Incubate the Erlenmeyer flask in the shaker overnight at 37 °C.
   4. Induce protein expression by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG) to the Erlenmeyer flask and continue the incubation for a further 2 h.
   5. To get cell pellets, centrifuge at 5,000 x g for 15 min. Remove the supernatant.
      NOTE: The cell pellets can be stored at -20 °C.
   6. Resuspend the cell pellets in 40 mL of PBS and sonicate 3 x 20 s on ice. Add 1 mM PMSF, 1 mM EDTA, and protease inhibitor cocktail (1 tablet) to prevent proteolysis.
   7. Centrifuge the suspension for 30 min 50,000 x g to remove cell debris and apply the supernatant onto 5 mL GST-trap column.
   8. After washing the column with 30 mL PBS, elute GST-FKBP51 and GST-FKBP52 with 5 mL of 10 mM glutathione in PBS.
   9. Concentrate proteins on 15 mL 10.000 MWCO centrifugation unit. To remove free glutathione, pass concentrates through PD-10 column equilibrated with 0.5x PBS and again concentrate on the filter centrifuge device.
   10. Collect protein-containing fractions. Verify the proteins in SDS-PAGE and adjust protein concentrations to 1 mg/mL.
       NOTE: Typical protein yield is 2-5 mg/L culture. The protein can be stored at -20 °C.

2. Coupling of Hsp90 C-terminal peptide to the acceptor beads (Figure 1B)

1. Hsp90 peptide preparation
   1. Synthesize ten amino acid peptide NH₂-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform (UniProt ID: P08238) by a peptide synthesis service.
   2. Dilute the Hsp90 peptide in PBS to 1 mg/mL concentration.
2. Acceptor beads preparation
   1. Dilute the unconjugated acceptor beads in PBS to 1 mg/mL concentration and transfer to a 1.5 mL tube.
   2. Perform the washing by centrifugation at 16,000 x g for 15 min. Carefully remove the supernatant.
3. Conjugation
   1. Set the ratio between beads and peptide as 10:1. In the 1.5 mL tube containing 1 mg of acceptor bead pellet (prepared as described above), add 1 mL of PBS (pH 7.4), 0.1 mg of diluted peptide, 1.25 µL of Tween-20, 10 µL of a 400 mM solution of sodium cyanoborohydride (NaBH₃CN) in water.
      CAUTION: NaBH₃CN is toxic; use a fume hood and gloves. NaBH₃CN solution should be freshly prepared.
   2. Incubate for 24 h at 37 °C with end-over-end agitation (10-20 rpm) on a rotary shaker.
4. Reaction quenching and beads washing
   1. Add 20 µL of 1 M Tris-HCl (pH 8.0) solution to the reaction to block unreacted sites. Incubate for 1 h at 37 °C.
   2. Centrifuge at 16,000 x g (or maximum speed) for 15 min at 4 °C. Remove the supernatant and resuspend the bead pellet in 1 mL of Tris-HCl solution (100 mM, pH 8.0).
   3. Repeat the washing step three times.
   4. After the last centrifugation, resuspend the beads at 1 mg/mL in storage buffer (1 mL of 0.5 × PBS with 0.01% sodium azide as a preservative). Store the conjugated acceptor bead solution at 4 °C light protected.
      CAUTION: Sodium azide is toxic; use a fume hood and gloves.

3. The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide, and inhibition with small molecular mass compounds (Figure 1C)

1. GST-tagged proteins interacting with glutathione donor beads
   1. Set up the reactions in 384-well plates.
   2. Prepare the solution containing 10 µg/mL of the glutathione donor beads in 0.5x PBS, pH 7.4.
      NOTE: After prolonged storage, beads settle down and need to be vortexed.
   3. Add GST-FKBP51 or GST-FKBP52 to a final concentration of 10 µg/mL.
   4. Incubate in the dark at 25 °C for 10 min.
      NOTE: At this step, GST-tagged proteins will interact with glutathione attached to the beads. For each well, 22.5 µL of this mixture will be used. The concentration of the binding partners must be determined empirically. Titrate GST-FKBP51 and GST-FKBP52 and choose the concentration that gives the best signal.
2. Compound addition
   1. Make serial dilutions of test compounds in DMSO.
      NOTE: The concentrations used are typically 10, 30, 100, 300, 1,000, and 3,000 µM.
   2. Add 0.25 µL of DMSO (negative control) or Hsp90 C-terminal peptide (positive control, 30 µM) or compounds in DMSO to the corner of each well of the plate. Use triplicates for every compound concentration.
   3. Add 22.5 µL of the solution containing glutathione donor beads with GST-tagged proteins to each well.
   4. Shake the plate gently with hand but thoroughly. Incubate in the dark at 25 °C for 15 min.
      NOTE: During this time, compounds will interact with the TPR domain at the Hsp90 C-terminal peptide binding site.
3. Acceptor beads addition
   1. Dilute the acceptor beads with attached Hsp90 C-terminal peptide to 100 µg/mL in 0.5x PBS.
   2. Add 2.25 µL of diluted acceptor beads to each well.
   3. Mix gently but thoroughly. Incubate in the dark at 25 °C for 15 min.
      NOTE: At this step, donor and acceptor beads are brought into proximity by the protein-peptide interactions. The final volume of the reaction mixture is 25 µL. Therefore, the final concentrations of compounds are ranging from 0.1 to 30 µM.
4. Plate reading
   NOTE: Read the plate using a plate reader set in the relevant mode.
   1. Turn on the instrument and open the software
   2. Choose the relevant protocol.
   3. Click Edit plate map and select the well being used in the plate for measurement.
   4. Click Next to continue and Run the selected protocol.
   5. After measurement, click Show Results to view results.
   6. Export the data.

Table 1: PCR reaction set up for human FKBP51 and FKBP52 DNA amplification.

Reaction component	Volume (µL)
PCR buffer (5 x concentrate)	4
Forward primer	1
Reverse primer	1
Plasmid	0.5
dNTP mix (10 mM each)	0.5
Phusion DNA polymerase	0.5
Water (DNA grade)	12.5
Total	20

Table 2: Thermocyler conditions for human FKBP51 and FKBP52 DNA amplification.

Stage	Temperature (°C)	Time	Cycles
Initial denaturation	94	3 min	1
Denaturation	94	30 sec	35
Annealing	56	30 sec
Extension	72	1 min
Final extension	72	5  min	1
Note: Lid temperature is 105 °C.

Resultados

Figure 1: Schematic of this protocol. (A) Expression and purification of GST-FKBP51 and GST-FKBP52. (B) Coupling of Hsp90 C-terminal peptide to the acceptor beads. (C) The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide. Inhibition with small molecular mass compounds.

Materiales

Name	Company	Catalog Number	Comments
384-well plates	Perkin Elmer	6008350	Assay volume 25 ml
Amicon 10.000 MWCO centrifugation unit	Millipore	UFC901008	Concentrate protein
Ampicillin	Sigma	A0166	Antibiotics
Bacteria shaker Unimax 1010	Heidolph		Culture bacteria
cDNA clones for human FKBP51	Source BioScience	clone id: 5723416	pCMV-SPORT6 vector
cDNA clones for human FKBP52	Source BioScience	clone id: 7474554	pCMV-SPORT6 vector
Chemically Competent E. coli	Invitrogen	C602003	One Shot BL21 Star (DE3)
DMSO	Supelco	1.02952.1000	Dilute compounds
DPBS	Gibco	14190-144	Prepare solution
EDTA	Calbiochem	344504	Prevent proteolysis during sonication
Glutathione	Sigma	G-4251	Elute GST-tagged proteins
Glutathione donor beads	Perkin Elmer	6765300	Donor bead
GST-trap column	Cytiva (GE Healthcare)	17528201	Purify GST-tagged proteins
Isopropyl-β-D-thiogalactoside	Thermo Fisher Scientific	R0392	Induce protein expression
LB Broth (Miller)	Sigma	L3522	Microbial growth medium
PCR instrument	BIO-RAD	S1000 Thermal Cycler	Amplification/PCR
PD-10 column	Cytiva (GE Healthcare)	17085101	Solution exchange
pGEX-6P-1 vector	Cytiva (GE Healthcare)	28954648	Plasmid
pGEX-6P-2 vector	Cytiva (GE Healthcare)	28954650	Plasmid
Plate reader	Perkin Elmer	EnSpire 2300 Multilabel Reader	Read alpha plate
Plate reader software	Perkin Elmer	EnSpire Manager	Plate reader software
Plate reader software protocol	Perkin Elmer	Alpha 384-well Low volume	Use this protocol to read plate
PMSF	Sigma	P7626	Prevent proteolysis during sonication
protease inhibitor cocktail	Sigma	S8830	Prevent proteolysis during sonication
Sodium azide	Sigma	S2002	As a preservative
Sodium cyanoborohydride (NaBH3CN)	Sigma	156159	Activates matrix for coupling
Ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform	Peptide 2.0 inc		Synthesize Hsp90 C-terminal peptide
Test-Tube Rotator	LABINCO		Make end-over-end agitation
Tris-HCl	Sigma	10708976001	Block unreacted sites of acceptor beads
Tween-20	Sigma	P1379	Prevent beads aggregation
Ultra centrifuge Avanti J-20 XP	Beckman Coulter		Centrifuge to get bacteria cell pellets
Ultrasonic cell disruptor	Microson		Sonicate cells to release protein
Unconjugated acceptor beads	Perkin Elmer	6762003	Acceptor beads
XCell SureLock Mini-Cell and XCell II Blot Module	Invitrogen	EI0002	SDS-PAGE