Purification of His-Tagged Glycoproteins

TIMING: 4–6 h

The produced recombinant glycoproteins can be highly useful for a variety of applications including studies on how a particular glycan feature modulates protein function or distribution in vivo or targeting of a glycan-binding receptor. For these experiments it is important to work with highly pure material. This step describes how glycoproteins can be purified from cell culture supernatant of the transfected isogenic cells.

Filter the cell culture supernatant containing secreted His-tagged glycoprotein through a 0.45μm vacuum filter system to remove any cell debris.

Pack about 200 μL nickel-nitrilotriacetic acid (Ni-NTA) affinity agarose into a 2 mL gravity-flow column and equilibrate the column with 5 agarose bed volumes (1 mL) equilibration buffer.

Mix 30 mL of the cell culture supernatant with 10 mL of 4x column equilibration buffer.

Run the sample twice over the Ni-NTA purification column and wash the column thrice with 10 agarose bed volumes (2 mL) column washing buffer.

CRITICAL: Keep the flow rate at a moderate speed, as fast flow will reduce the yield of glycoproteins.

Optional: Collect the flow-through to assess the purification efficiency by SDS-PAGE analysis or other methods. The column size can be scaled up depending on the sample volume. Read the manufacturer’s instructions regarding the binding capacity of the Ni-NTA agarose (usually up to 50 mg/mL of 6x His-tagged proteins), imidazole concentrations and buffer volumes required. Alternatively, instead of using gravity-flow, Ni-NTA agarose beads can be added to the supernatant in 50 mL tubes followed by 12–18 hrs incubation at 4˚C and spin purification. This method may increase yield, but reduces purity with more non-specific proteins purified.

Elute the immobilized protein by adding 2 agarose bed volumes (0.4 mL) column elution buffer and collect into a 1.5 mL tube. Repeat this step two times to obtain a total of three fractions in separate tubes.

Determine the protein content in the fractions containing the His-tagged protein by SDS-PAGE analysis.

Select the fraction with the highest protein content (or pool fractions) and exchange the elution buffer to MQ, 1x PBS, 50 mM ammonium bicarbonate (Ambic) or another buffer type (compatible with the planned downstream applications) using a desalting column (e.g. PD MiniTrap™ G-25 column or Zeba™ column) according to the manufacturer’s instructions.

Note: Ambic buffer is suitable for further mass spectrometry analysis.

Aliquot the purified glycoprotein and store at -20°C or flash freeze the protein and store at -80 °C, which may preserve protein integrity better.

Optional: Correct glycosylation of the purified recombinant glycoproteins can be confirmed by mass spectrometry.