Recombinant protein expression in E.coli 72

The genes of interest were cloned into pGEX-6p-2 vector (Pharmacia, USA), which contains the DNA sequence encoding GST and allows in-frame fusion of protein of interest and GST. The resulting plasmids were transformed into the BL21 E.coli strain.

One single colony was selected, and after verification via PCR amplification, the colony was inoculated into 3 ml of LB medium supplemented with ampicillin and incubated at 37°C overnight with vigorous shaking. On the next day, the overnight cell culture was transferred to 200 ml of LB liquid plus ampicillin and grown until the OD₆₀₀ value reached 0.6-1.0. The protein of interest fused with GST was induced at 16°C by the supplement of Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, and incubated for 12-24 hr. The cells were then harvested by centrifugation at 3,000 rpm for 20 min.

The cell pellets from the previous step was lysed and purified as described below. The cells were resuspended in 35 ml of pre-chilled lysis buffer (10 mM Tris pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.5 mM DTT and 1% Triton X-100). The cell suspension was then subjected to sonication to disrupt the cells, followed by centrifugation at 10,000 rpm for 20 min. The cell lysate was then transferred to 50 ml Falcon tube and mixed

with 250 μl glutathione sepharose beads (Amersham Biosciences, USA). This solution was incubated at 4°C with rotation for 2-3 hr to allow the GST-tagged protein to bind to the glutathione sepharose beads. Subsequently, the beads were pelleted by centrifugation at 1,000 rpm for 5 min. After discarding the supernatant, the beads were washed with the cell lysis buffer for at least three times. After the final wash, the beads were analyzed by SDS-PAGE gel electrophoresis followed by staining with Comassie blue (sections 2.9.4 and 2.9.5). The beads can be kept at 4°C up to several weeks.

2.9.2 Expression of protein of interest using in vitro translation system

The synthesis of protein of interest was performed with TNT T7 Quick Coupled Transcription/Translation System (Promega, USA) according to the manufacturer’s instructions. The genes of interest were cloned into the pGADT7 or pGBKT7 vectors, which contains the DNA sequence of HA or MYC epitope tag, respectively, which allows the detection of protein translated via western blot. The pGBKT7 vector was directly used in the TNT system, while the pGADT7 vector was firstly linearized before subjected into the TNT system. The in vitro transcription/translation reaction was set up by mixing the 1 μg plasmid DNA template with 40 μl TNT Quick Coupled Transcription/Translation Master Mix, 1 pmol methionine and topped up to 50 μl with nuclease-free water. The reaction was incubated at 30°C for 90 min. The protein translated can be used immediately or stored at -80°C.

Figure 3. Diagram shows the process of in vitro GST pull-down

2.9.3 In vitro pull-down assay

The two proteins, one immobilized on the GST beads (30 μl) from section 2.8.1 and the other synthesized from in vitro-translation system, were mixed together in 1 ml of pre-colded IP buffer (50 mM Hepes pH 7.5, 150 mM KCl, 5 mM MgCl₂, 10 μM ZnSO4, 1% Triton X-100 and 0.05% SDS) and incubated at 4°C for 2-3 hr with rotation. Subsequently, the beads were precipitated by centrifugation at 2,500 rpm for 1 min at 4°C. The beads were then washed with 1 ml IP buffer at 4°C for 10 min with rotation, and precipitated again. The previous step was repeated for another three to four times to remove any non-specific binding. The beads were then used for SDS-PAGE gel electrophoresis and the fusion protein was detected with western blot. For this in vitro pull-down assay, GST protein immobilized on the GST beads (30 μl) was used as the negative control and subjected to the same experimental process as the GST fusion proteins.

2.9.4 SDS-PAGE gel electrophoresis

SDS-PAGE gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis gel electrophoresis) is a technique commonly used for separating proteins according to the elctrophoretic mobility, such as molecular weight, post-translational modifications.

Prior to the electrophoresis, the PAGE gel was set up and assembled. The SDS-PAGE gel is comprised of a separating gel on the bottom and a stacking gel on the top.

The separating gel was set up by mixing appropriate amount of 30% polyacrylamide, 1.5M Tris pH 8.8, 10% SDS, distilled water, 10% ammonium persulfate (AP) and TEMED. The solution was mixed thoroughly and immediately loaded into the gel

caster. After the separating gel was polymerized, the stacking gel was set up by mixing appropriate amount of 30% polyacrylamide, 0.5M Tris pH 6.8, 10% SDS, distilled water, 10% AP and TEMED, and loaded into the gel caster on top of the separating gel. The comb was placed to create the wells. After the polymerization of the stacking gel, the SDS-PAGE gel was ready to use.

To prepare the protein samples for electrophoresis, appropriate amount of 6X SDS sample loading dye (125mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 0.2%

bromophenol blue) was added into the protein samples, and mixed thoroughly. The protein samples were subsequently denatured by boiling at 100°C for 10 min and kept on ice.

The SDS-PAGE gel was assembled into the electrophoresis apparatus, and 1 X SDS running buffer (25 mM Tris, 196 mM glycine and 0.1% SDS) was added. The protein samples and Precision Plus Protein^TM standards All blue maker (Biorad, USA) were loaded into wells of the SDS-PAGE gel. Subsequently, the gel was run at 30 mA.

After electrophoresis, the gel was stained with Coomassie blue or continued with western blot depending on different purposes of the experiments.

2.9.5 Coomassie blue staining

After electrophoresis, the gel was placed in the staining solution (0.1% Coomassie Brilliant Blue R-250 dissolved in 10% acetic acid, 20% methanol and 70% distilled water), and incubated at room temperature for 20-60 min with shaking. The staining solution was then replaced by the destaining solution (10% acetic acid, 20% methanol

and 70% distilled water) and incubated at room temperature with shaking until the protein bands were visible.