This method allows to study formations of protein complexes that require co-expression for efficient test samples, such as the formation of heterodimers. Main end potential of this method is exploiting the co-expression of differentially-tagged studied proteins using combinations of compatible vectors to promote efficient complex assembly, along with time-efficient workflow and scalability allowing rapid patch-testing of multiple interactions. This method can also be used for rapid screening of optimal protein expression and purification conditions.
Visual demonstration helps correctly perform critical steps of the protocol such as co-expression setup, cell disruption, and subsequent pulldown steps. To begin, grow Escherichia coli BL21(DE3)strain in Luria-Bertani, or LB, media at 37 degrees Celsius to an optical density, or OD, of 0.1 to 0.2. Centrifuge one milliliter of the bacterial suspension for one minute at 9, 000 G at four degrees Celsius and discard the supernatant.
Then, add 0.5 milliliters of ice-cold transformation buffer, or TB, and incubate for 10 minutes on ice. At the end of incubation, centrifuge for 30 seconds at 8, 000 G at four degrees Celsius and discard the supernatant. Then, add 100 microliters of TB buffer, 100 nanograms of each vector, and incubate for 30 minutes on ice.
Heat at 42 degrees Celsius for 150 seconds, then chill on ice for one minute. Next, add one milliliter of LB media without antibiotics and incubate at 37 degrees Celsius for 90 minutes. After incubation, centrifuge and resuspend the pellet in 100 microliters of LB media before plating the suspension on an LB agar plate containing 0.5%glucose and corresponding antibiotics.
Incubate the plate overnight at 37 degrees Celsius. The following day, flush the cells from the plate with two milliliters of LB media into 50 milliliters of LB media with corresponding antibiotics. Add metal ions or other known co-factors before storing an aliquot with 20%glycerol at minus 70 degrees Celsius for subsequent experiments.
Grow the cells with a constant rotation of 220 RPM at 37 degrees Celsius to an OD of 0.5 to 0.7. Cool the culture to room temperature and add IPTG to a final concentration of one millimole. Store a 20-microliter aliquot of cell suspension as a control of the uninduced sample.
Incubate the cells overnight at 220 RPM at 18 degrees Celsius. The next day, divide the bacterial suspension into two parts and store 20-microliter aliquots to confirm protein expression. Centrifuge the remaining cell suspension at 4, 000 G for 15 minutes.
For pulldown assay, resuspend the pellets in one milliliter of ice-cold lysis buffer with protease inhibitors and reducing agents added immediately prior to the experiment. Adjust the buffer composition for the tested proteins. Disrupt the cells by sonication on ice and store a 20-microliter aliquot for electrophoresis.
Centrifuge at 16, 000 G for 30 minutes and collect 20 microliters of the clarified lysate for electrophoresis. Equilibrate the resin with one milliliter of ice-cold lysis buffer for 10 minutes, then centrifuge at 2, 000 G for 30 seconds and discard the supernatant. Add cell lysates to the resin and incubate for 10 minutes at a constant rotation of 15 RPM.
Then, centrifuge and discard the supernatant before collecting 20 microliters of the unbound fraction for analysis. Next, add one milliliter of ice-cold wash buffer and incubate for one minute. Again, centrifuge and discard the supernatant.
Then, perform two long washes with ice-cold wash buffer. After the second wash, elute the bound proteins with 50 microliters of the elution buffer in a shaker at 1, 200 RPM at four degrees Celsius for 10 minutes. Analyze the eluted proteins with SDS polyacrylamide gel electrophoresis.
Studies of the zinc finger-associated domain, or ZAD, homo-dimerization in maltose-binding protein, or MBP, and 6xHistidine pulldown assays are shown here. The co-expression of MBP and Thioredoxin 6xHistidine-fused ZADs shows good and reproducible homodimerization activity, appearing as an additional band in the SDS-PAGE results of the MBP pulldown assay. Another example of using the method to study alternative complex formation is shown here.
In the co-expression assay, 6xHistidine-tagged ENY2 interacted both with GST-tagged Sgf11 and MBP-CTCF, but no GST-Sgf11 was present in the MBP pulldowns and vice-versa. A step-by-step comparison of required time intervals in the workflow of the conventional pulldown assay compared to pulldown coupled to co-expression is shown here. The results of utilizing both techniques to study the same interaction between the BTB domain of the CP190 protein and the GST-tagged C-terminal domain of Drosophila CTCF protein are shown here.
The correct choice of affinity and solubility test, Thioredoxin, GST, or MBP, is critical for the efficient execution of the assay. Another critical step is fast and uniform cell disruption. It's highly recommended to use multi-tip sonicator probes.
This method allow the direct study of the heterodimer formation between protein domains that otherwise form homodimers that would not disassociate during the incubation of purified proteins in conventional pulldown assay.
