Interaction cloning experiments
Interaction cloning experiments
Interacting cloning (also known as expression cloning) is used to identify and clone proteins encoding proteins that interact with a target or 'bait' protein. This approach bypasses the necessary processes of purification, microsequencing or antibody preparation. Source The Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
Interacting Clones
Principle
It requires a gene encoding a decoy protein and an appropriate expression library constructed with a phage expression vector such as λgt 11. The gene encoding the bait protein is commonly used to synthesize recombinant fusion proteins in. Recombinant fusion proteins are labeled with radioactive [32P]. Since the recognition site of the cAMP-dependent protein kinase (protein kinase A, PKA) is introduced into the recombinant fusion protein, it can be enzymatically phosphorylated by PKA and [γ-32P]ATP. This tagged protein can be used as a probe to screen cDNA expression libraries derived from λ phage that express the β-galactosidase fusion protein in the reading frame. The phage lyses the cell to form a phage spot and releases the fusion protein, which is adsorbed onto a nitrocellulose filter membrane, which is sealed with an excess of non-specific protein to exclude non-specific binding, and then detected by the radiolabeled bait protein.
Materials and Instruments
cAMP-dependent protein kinase Purified GST-decoy protein fusion (with PKA recognition site) E.coli Y1090 r- or other appropriate host bacteria Move 1. Resuspend 250 U/μl PKA in 25 μl of freshly prepared 40 mmol/L DTT and allow to warm up at room temperature for approximately 10 min before use. PKA can be temporarily stored at 4°C, but the activity can only be maintained for 2~3 days. 2. 2. Prepare a mixture for the phosphorylation reaction containing the following components: 1 μl 10 U/μl PKA (obtained from step 1) 3 μl 10 × PKA buffer 5 μl 10 mCi/ml (6000 mCi/mmol) [ γ-32P ] ATP 1~10 μl (approx. 1 μg) purified GST-bait protein fusion Add H2O to 30 μl Incubate at room temperature for 1 h. Fusions that are not linked to the bait protein but contain a PKA recognition site should also be expressed as a control to determine whether the observed interaction is specific to the bait protein. It is preferable to label both the bait protein and the fusion that is not linked to the bait protein (in separate reactions). 3. 3. Add 170 μl of ice-cold Z'-KCl (to abort the reaction) and keep on ice. 4. pour the Sephadex G-50 equilibrated in Z'-KCl into a 3 ml disposable plastic column (or a 3 ml glass wool stoppered syringe) with a final bed volume of approximately 3 ml. allow the buffer in the column to flow out to the level of the top of the column bed. 5. Spike all of the phosphorylated reactants (obtained from step 3) into the column and collect the effluent in a 1.5 ml centrifuge tube. In a second tube, add a 200 μl portion of Z'-KCl and collect the effluent. Repeat the addition and collection steps 10 more times to collect a total of 12 200 μl portions of effluent. 6. 6. Using a scintillation counter, measure the Cherenkov number to determine the effluent containing the highest specific activity and calculate cpm/μl. Typically, two strong signal peaks are seen: the first peak is between the 5th and 9th effluent and is generated by the labeled protein. The second peak is discarded, and is generated by unbound ATP. 7. Optional: Analyze a small amount (1-2 μl) of 32P-labeled protein probe by SDS-PAGE and autoradiograph. Typically, two strong signal bands are seen: one at the expected molecular mass size of the GST-decoy protein fusion, and the other at the expected molecular mass size of the non-fused GST (28 kDa). This is due to the fact that in fusion proteins the GST binding site to the bait protein is itself sensitive to protease cleavage. The presence of labeled GST proteins in the probe should not interfere with the screening; control experiments with labeled GST should be added at subsequent screening stages. 8. Make serial dilutions to determine the titer of the phage cDNA library. 9. 9. Overnight culture 50 ml of E.coli Y1090 r- (or other suitable host bacteria) in LB medium containing appropriate selective antibiotics, 10 mmol/L MgSO4 and 0.2% maltose. Centrifuge the bacterial cells at 2000 g for 10 min at room temperature and resuspend the bacterial cells with 25 ml of 10 mmol/L MgSO4. 10. 10. Soak nitrocellulose filter membranes in 10 mmol/L IPTG for 15 min at room temperature and air dry. 11. 11. Prepare eight 1.5 ml centrifuge tubes, each containing 600 Y1090 r-cells (from step 9) and approximately 40,000 pfu of phage (from step 8), and float for 15 min at 37°C. Pour the contents of each tube into a tube containing 7 ml of 0.7% top agarose at 47°C and quickly pour onto a 150 mm LB plate (or antibiotic-containing LB plate, if desired). (or LB plates with antibiotics if desired). Incubate the plate at 42°C for about 3 h until visible microphages develop. 12. 12. Spread the pre-saturated filter membrane with IPTG solution onto the plate and continue to incubate at 37°C for 6-8 h. Cool the plate at 4°C for 15 min (or overnight). The absence of induction of the lacZ gene promoter during phage plaque formation ensures that none of the library-encoded proteins that are toxic to phage growth are expressed during phage plaque formation. Usually incubate with IPTG-impregnated filter membranes for 6-8 h. For convenience, incubate overnight with IPTG-impregnated filter membranes. 13. Mark the orientation of each membrane with several punctures with a 22-G needle dipped in India ink. Remove the membranes from the plate and wash with TBS-T at room temperature with shaking for 15 min. soak the membranes in 100 ml of HBB and shake at 4°C for 1 to 4 h (or overnight). 14. 14. Soak the membranes in BB containing 2.5 × 105-5 × 105 cpm/ml isotope-labeled fusion protein (prepared from step 6) and shake at 4°C overnight. All 8 membranes can be placed in a 150 mm dish and immersed in 30-40 ml of probe solution. Seal the dish with paraffin film and place it in a Plexiglas box for shielding. Alternatively, the membrane and solution can be placed in a sealed bag. The probe solution can be stored at 4°C and used repeatedly for secondary and tertiary screening. 15. 15. Wash the membrane 3 times at room temperature in 100 ml BB with shaking for 10 min each time, air-dry and press. 16. Using the larger end of a Pasteur pipette, remove the agarose block by inserting it at the site of the positive clone. Place in a 1.5 ml microcentrifuge tube and add 1 ml SM and a drop of chloroform. The titer is determined by serial dilution and serial screening is performed to obtain purified clones. Agarose blocks can be stored at 4°C for several months. Therefore, if many false-positive clones are identified in the primary screen, all of them can be saved for later analysis if needed. The next screen is performed on 100 mm LB plates with about 2000 pfu/plate for the secondary screen and about 300-500 pfu/plate for the tertiary screen. Before homogeneous clones are obtained (usually during tertiary screening), a control experiment should be performed to exclude clones that may interact with the GST fusion portion of the probe. This can be done by cutting the membrane in half and probing one half with the labeled GST-target protein fusion and the other half with the labeled GST or unlabeled control GST fusion protein. Caveat Radiolabeling is used in this protocol and should be handled with care and appropriate shielding protection. For more product details, please visit Aladdin Scientific website.
DTT PKA buffer [γ-32P] ATP Z'-KCl Sephadex G-50 LB medium MgSO4 IPTG Top layer agarose Tris buffer salt solution (with Triton X-100) India ink HEPES Closure buffer (HBB) Binding buffer (BB) Suspension medium (SM) Chloroform
3 ml disposable plastic column/disposable syringe and glass wool Scintillation counter Scintillation solution Benchtop centrifuge Nitric acid fiber filter membrane 22-G needle