2.6. Purification of heterologously expressed protein from extracellular media

Prepare three buffers for purification of recombinant proteins. All buffers used for chromatography must be filtered and degassed. Filter solutions using a setup comprised of a coarse porosity fritted glass filter support base (Wheaton) topped with a regenerated cellulose membrane filter (47 mm, 0.45 μm pore size, Whatman) clasped with an aluminum clamp (Wheaton) to a 500 mL glass funnel (Wheaton) that is fitted onto a 1L vacuum flask attached to vacuum source via a non-collapsible rubber hose. Transfer buffer to upper glass funnel and filter into the vacuum flask by applying low vacuum. Remove filtration device, add a stir-bar, seal top of flask with rubber stopper, attach vacuum hose to sidearm of vacuum flask, turn on vacuum to low, and stir solution on a stir plate (100rpm) to promote release of gases for 60 min to overnight. Degassing is complete when it no longer appears to “boil.” Chill buffers to 4 °C prior to use.

10 × Buffer A: 500 mM HEPES free acid, 4M NaCl, and 200 mM Imidazole, pH 7.2

Buffer A: 50 mM HEPES free acid, 400 mM NaCl, and 20 mM Imidazole, pH 7.2

Buffer B: 50 mM HEPES free acid, 400 mM NaCl, and 500 mM Imidazole, pH 7.2

Note: Proper preparation of buffers is critical for chromatography experiments. Dissolved gases in buffers can come out of solution (outgas) and form bubbles within the resin bed, interfering with column function and resulting in decreased binding capacity and elution efficiency.

Harvest extracellular medium 6 days post-transfection by sequential centrifugation at 377 × g for 15 min, 2683 × g for 15 min, and 22,100 × g for 30 min at 4°C to remove any remaining cellular debris and larger aggregates. Coming® 250 mL polypropylene (PP) centrifuge tubes or equivalent are ideal for applications requiring large volume centrifugation. All samples must be kept on ice.

Measure the total volume of supernatant, and slowly add 10 × Buffer A to reach a final concentration of 1 ×. For example, for 90mL of cell culture supernatant, 10mL of 10 × Buffer A would be added to reach a final concentration of 1 ×, which is referred to as buffer-adjusted extracellular media. Pass buffer-adjusted extracellular media through a 5-μm cellulose filter (Pall Corporation, https://www.pall.com) followed by a 0.45-μm filter (Pall Corporation, https://www.pall.com).

Note: This filtration step is critical to avoid damaging or locking up chromatography columns in the next steps.

IMAC1 purification: Carry out small scale purification of 8 × -His-tagged fusion proteins from buffer-adjusted extracellular media with a HisTrap HP (1 mL column volume (CV); GE Healthcare) prepacked column on an ÄKTA Pure 25 L protein purification system (GE Healthcare) using a step gradient. Maintain the flow rate at 1 mLmin⁻¹ throughout the purification. If large scale purification is required, a HisPrep FF 16/10 column (20 mL CV; GE Healthcare) can be used by adjusting the protocol scheme according to the recommended CV values with flow rates of up to 4mLmin⁻¹ (recommended). To eliminate the possibility of protein contamination, purification of different enzymes or enzyme variants should be carried out on individual columns.

Note: For new columns, it is important to perform a blank run to remove any weakly bound Ni²⁺ ions. Wash columns in line at 1 mL min⁻¹ beginning with 5 CV of water, followed by 5 CV of 100% Buffer A, followed by a linear gradient of Buffer A to 100% Buffer B for 5 CV. Then, wash the columns with 100% Buffer B for 5 CV, followed by a linear gradient of 100% Buffer B to 100% Buffer A in 5 CV, prior to re-equilibrating with Buffer A.

Note: Columns can be washed with water containing 0.04% (w/v) sodium azide and stored at 4 °C in 20% ethanol between uses.

For purification, all steps are carried out at 1 mL min⁻¹. Load adjusted media onto a HisTrap HP column pre-equilibrated with at least 5 CV Buffer A. Wash and elute the column with a step gradient of 5 CV per condition, consisting of three sequential wash steps of 0%, 10%, and 20% Buffer B, followed by two elution steps of 60% and 100% Buffer B (Fig. 5). Fractions containing GFP fluorescence (typically 60% Buffer B elution fractions), which will also be visibly green in natural light, represent those containing the target fusion protein.

Flow chart for protein purification on a His Trap HP (1 mL).

After purification, take 30 μL aliquots of adjusted media, flow-through, wash, and eluted fractions and dilute each with 10 μL of 4 × Laemmli sample buffer (Bio-Rad) containing reducing agent. Heat the mixed samples at 95 °C for 6 min and centrifuge at 12,500 × g for 2 min before performing SDS-PAGE to assess protein purity (Fig. 6).

IMAC1 purification of XyG glycosyltransferases. AKTA UV chromatogram (left) and SDS-PAGE of the eluted fractions to assess protein purification (right). Color code: unbound protein (red), wash (green), wash with 10% Buffer B (purple), wash with 20% Buffer B (light blue), elution with 60% Buffer B (orange), elution with 100% Buffer B (dark blue). UV is shown in milli-absorbance (mA). EM, FT, W1, E1, E2, and M represent extracellular media, flow through, wash (20% of Buffer B), elution (60% of Buffer B), elution (100% of Buffer B) and marker, respectively.

Dialysis and buffer exchange: Further concentrate eluted protein from the IMAC1 step to approximately 2mg/mL using a 5 mL Amicon concentrator with a 10-kDa molecular weight cutoff. At this point, if necessary, the protein can be further purified using the size exclusion chromatography protocol described in step 5 in Section 2.7 or exchanged into a new buffer for biochemical analysis or storage in step 8.

Exchange the protein buffer in 75 mM HEPES sodium salt pH 6.7 via dialysis (3.5 kDa MWCO) overnight in 500mL of cold buffer at 4°C stirring at 100rpm. Decant buffer and replace with 500 mL of fresh cold (4°C) buffer and continue dialysis for an additional 2–4h an additional two times. Dialyzed samples are ready to use for assays or flash frozen.

Note: Buffer conditions for dialysis and storage must be empirically determined for each protein.

Note: Do not use regenerated cellulose dialysis membranes when working with glycoside hydrolases that cleave β−1,4-glycosidic linkages.