View - ResearchGate

160 Godat et al.
the above protocols to include the appropriate amount of denaturant (up to 6 M
guanidine–HCl or up to 8 M urea) in binding, wash and elution buffers.
3.6. Purification of HQ-Tagged Proteins Expressed in Insect
and Mammalian Cells Using Magnetic Ni Particles
Bacterial expression of recombinant His-tagged proteins is a common
technique. However, insect cells and mammalian cells are becoming more
widely used expression systems for expression of recombinant proteins. These
eukaryotic expression systems may allow more natural processing and modification
of recombinant proteins, which are not possible in bacterial expression
system. HQ tag can also be used in these expression systems.
3.6.1. Preparation of Insect and Mammalian Cells
Insect or mammalian cells can be cultured under normal conditions. Process
cells at a cell density of 2 × 10 6 cells/ml of culture. Adherent cells may be
removed from tissue culture plastic by scraping and resuspending in culture
medium to this density. Cells may be processed in culture medium containing
up to 10% serum. Processing more than the indicated number of cells per
1 ml sample may result in reduced protein yield and increased non-specific
binding (10).
3.6.2. Purification of Intracellular Expressed HQ-Tagged Proteins
from Cultured Insect or Mammalian Cells
1. Add 110 μl of FastBreak Cell Lysis Reagent, 10×, to 1 ml of insect or
mammalian cells in culture medium (see Note 7).
2. Add 1 μl DNase I (see Note 3) to the lysed insect or mammalian cell culture.
3. Incubate with shaking for 10–20 min at room temperature on a rotary mixer or
shaking platform.
4. Vortex the MagneHis Ni Particles to a uniform suspension (see Note 4).
5. Add 30 μl of the MagneHis Ni Particles to 1.1 ml of cell lysate.
6. Add 1 M imidazole (pH 8) to a final concentration of 20 mM to decrease
non-specific binding of serum proteins (22 μl of 1 M imidazole per 1.1 ml of
sample).
7. Invert tube to mix (˜10 times) and incubate for 2 min at room temperature.
8. Place the tube in the appropriate magnetic stand for approximately 30 s to capture
the MagneHis Ni Particles. Using a pipette, carefully remove the supernatant.
9. Remove the tube from the magnetic stand. Add 500 μl of MagneHis
Binding/Wash Buffer containing 500 mM NaCl to the MagneHis Ni Particles
and pipette to mix. Make sure that the particles are resuspended well.