Editing Protein Expression 293 P2

Protocol<br/>NOTE: The protocol is suitable for any scale of expression, therefore volumes and quantities of reagents should be scaled proportionally. A suitable mammalian expression vector must be used for this protocol. Here we used a modified pcDNA 3.1 expression vector that conveniently allows the inclusion or exclusion of an affinity tag depending on the choice of restriction enzymes used in the cloning (Figure 1). The following protocol describes a large scale transfection.

1. Large Scale Culture/Transfection in 1 L Roller Bottles

Grow and maintain HEK293F suspension-adapted cells according to standard protocols. Typically starter cultures of between 30 and 100 ml are grown in 250 ml conical cell culture flasks.

Seed cells at 0.5 x 106 cells/ml into a final volume of 300 ml in each 1 L roller bottle. NOTE: Roller bottles accommodate a minimum of 150 ml to a maximum of 300 ml of suspension culture. For larger scale transfections use multiple bottles.<br/>Incubate for 24 hr in an orbital shaker incubator at 37 °C, 120 rpm, and 5% CO2 until cells reach a density of 1.0 x 106 cells/ml (cells should divide approximately every 24 hr).<br/>Pipette a total of 300 µg of filter-sterilized DNA (see supplementary method) into 30 ml of PBS and vortex vigorously for 3 sec. NOTE: Use a total of 1 µg of DNA per million transfected cells. If two or more plasmids are to be co-transfected reduce the amount of each so that the ratio of DNA to cells remains constant.&nbsp;<br/>Add 1.2 ml of 0.5 mg/ml filter-sterilized PEI to the PBS/DNA solution and vortex vigorously for 3 sec.<br/>Incubate the mix at RT for 20 min.<br/>Add the DNA/PEI mix to the cells – which should be at a density of 1 x 106 cells/ml (step 1.3).<br/>Following co-transfection, incubate the cells in an orbital shaker incubator for a further 48 hr at 37 °C, 120 rpm, and 5% CO2.<br/>Harvest intracellular proteins by centrifuging cells at 3,000 x g for 5 min and store the pellet at -80 °C. NOTE: Alternatively, use a standard (non-CO2) shaking incubator, if the atmosphere above the culture is replaced with 5% CO2 and the bottle lid is sealed. The atmosphere will need to be replaced at each passage (normally every 2 days).<br/>2. Protein-complex Purification from Whole Cell Extract

This protocol is optimized for the purification of nuclear complexes using flag-tagged proteins.

Defrost cell pellet into ~40 ml of pre-chilled lysis buffer (100 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol, 0.3% Triton X-100, protease inhibitors) per L of culture.<br/>Re-suspend the pellet by pipetting up and down several times (to avoid foaming, do not vortex).<br/>Thoroughly re-suspend cells using a glass homogenizer. Sonicate for 3 cycles (15 sec on, 15 sec off). Centrifuge at 30,000 x g for 25 min at 4ºC and retain supernatant.<br/>Equilibrate 1.25 ml of anti-flag packed agarose resin per liter of culture by washing three times with resin equilibration buffer (100 mM potassium acetate, 50 mM Tris pH 7.5).<br/>Incubate the whole cell extract from step 2.3 with the affinity resin in one or more 50 ml centrifuge tubes and gently rotate the sample for 30-120 min at 4 °C.<br/>Centrifuge at 3,000 x g for 1 min at 4 °C, discard supernatant.<br/>Wash the resin with 45 ml of pre-chilled buffer 1 (100 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol, 0.3% Triton X-100). Centrifuge at 3,000 x g for 1 min and discard supernatant.<br/>Repeat step 2.7 using high salt buffer (300 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol), followed by low salt buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol) and TEV Cleavage Buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 0.5 mM TCEP). NOTE: Ensure each wash is brief i.e., sufficient to fully re-suspend the resin and no longer.<br/>Collect a 10 µl sample of resin and dilute into 1 volume of 2x protein loading buffer for analysis (bound protein control). Do not use reducing agents in order to avoid releasing the antibody from the resin.<br/>Re-suspend the resin into 8-10 ml of pre-chilled TEV cleavage buffer and transfer to a 15 ml centrifuge tube.<br/>Add ~40 µg of TEV protease (from stock at 1 mg/ml) per L of original culture and mix well by pipetting up and down several times.<br/>Replace tube atmosphere with 100% N2 gas to prevent protein oxidation.<br/>Gently rotate O/N at 4ºC.<br/>Centrifuge at 3,000 x g for 10 min. Transfer the supernatant into an ultra centrifugal filter with an appropriate molecular weight cut-off and concentrate down to 500 µl.<br/>Collect a 10 µl sample of the concentrated protein and dilute into 1 volume of 2x protein loading buffer for analysis. (TEV eluate control).<br/>Collect a 10 µl sample of the resin and dilute into 1 volume of 2x protein loading buffer for analysis. Do not use reducing agents in order to avoid releasing large amounts antibody from the resin. (Post-TEV resin control).<br/>Equilibrate the size exclusion chromatography column with gel filtration buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 0.5 mM TCEP).<br/>Filter the protein through a 0.22 µm filter<br/>Load the sample into the column and collect the fractions.<br/>Run samples from steps 2.9, 2.15 and 2.16 and the gel filtration fractions from step 2.19 on an SDS-PAGE and Coomassie stain for analysis. NOTE: It is advisable to run an SDS-PAGE of samples from steps 2.9, 2.15 and 2.16 prior to gel filtration to confirm expression of the target protein.
[[Category:Antibodies]]