Characterization of decoy proteins
Characterization of decoy proteins
The first step in capturing interacting proteins is to construct a decoy protein, which is a fusion of LexA to the target protein. A series of control experiments are performed to characterize whether the constructed decoy protein is suitable and needs to be modified or not.
Operation method
basic program
Materials and Instruments
Protein Move I. lacZ activation test1. Using standard subcloning techniques, the DNA encoding the target protein is inserted into the multiple junction of pEG202 to construct a fusion protein that conforms to the reading frame. For more product details, please visit Aladdin Scientific website.
CM medium
Water baths Incubators Electrotransferometers
2. The following combinations of plasmids were used to transform Saccharomyces cerevisiae EGY48 according to the " lithium acetate transformation method ".
(1) pBait+pSH18-34 (experimental group)
(2) pSH17-4+pSH18-34 (activated positive control group)
(3) pRFHM1+pSH18-34 (activation negative control group)
3. The transformation mixtures were spread on Glc/CM-Ura-His Spare Component Medium plates and incubated at 30°C overnight to select yeast containing both plasmids.
4. Pick at least 5 or 6 single colonies of each of the three transformants (experimental group, positive control and negative control) obtained in the previous step and place each clone on a master plate of Glc/CM-Ura-His Spare Component Medium.5. Place a piece of nylon membrane gently on the plate containing yeast, remove it after it is completely wetted, dry the nylon membrane in air for 5 min, turn the colony-stained side up and freeze it at -70°C for 10 min.
6. Take a piece of Whatman 3MM filter paper soaked in 1 x Z buffer containing 1 mg/ml Xgal and place the nylon membrane on it, colony side up. Alternatively, place the nylon membrane directly in the culture blood containing approximately 2 ml of Z buffer containing 1 mg/ml Xgal, incubate at 30°C and observe the color change of the clone.II. Inhibition test7. Transform Saccharomyces cerevisiae EGY48 with the following combinations of plasmids.
(1) pBait+pJK101 (experimental group)
(2) pRS423+pJK101 (inhibition test negative control group)
(3) pRFHM1+pJK101 (inhibition test positive control group)
8. Apply each transformation mixture to a plate of Glc/CM-Ura-His Spare Component Medium in order to select yeast cells with a pair of transforming plasmids, and incubate at 30°C until colonies appear, which usually takes 2 to 3 days, but not more than 4 days.
Figure 1. pEG202 LexA-fusion plasmid
9. Single colonies grown after transformation were streaked on plates of Glc/CM-Ura-His Spare Component Medium and incubated at 30°C overnight.10. Detect β-galactoside activity in three groups of transformed yeast strains (experimental, positive control, control) by liquid assay test (using Gal/CM Save Component Medium), by replica membrane analysis, or by a combination of the two assays. Detect β-galactosiduronic acid activity in three groups of transformed yeast strains (experimental group, positive control group, positive control group).III. LEU 2 experiment11. Take a single colony of Saccharomyces cerevisiae EGY48 transformed with the pBait and pSH18-34 reporter plasmids, disperse it in 500 μl of sterile water, take out 100 μl of it, dilute it in 1 ml of sterile water, and serially dilute it up to 1,000 times in sterile water at a 1/10 ratio.12. 100 μl of each dilution was spread on Glc/CM-Ura-His Spare Component Medium Plates and Glc/CM-Ura-His-Leu Spare Component Medium Plates, respectively, and incubated at 30°C overnight.13. Select clones that can be used as interaction traps for library selection; suitable pBaits should be unable to activate the lacZ and leuZ reporter genes, and are required to produce β-galactosidase activity that is lower than that of JK101+pRS423 and comparable to that of JK101+RFHM1. It may be appropriate for pBaits to have a low activation activity, but if pBaits have a strong activation activity, they should be truncated. If pBaits have a strong activation activity, they should be truncated.