Tao-Hsin Chang^{1, 2, a} and E. Yvonne Jones¹
¹Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of
Oxford, Oxford, OX3 7BN, UK
²Current address: Department of Molecular Biology and Genetics, Johns Hopkins University
School of Medicine, Baltimore, MD 21205, USA
^aTo whom correspondence may be addressed. E-mail: taohsin.chang@gmail.com

Purpose:
The protocol presented here has been used for the protein production of Frizzled4 cysteine-rich
domain (Fz4CRD), Fz7CRD, and Fz8CRD in HEK293 cells, using the transient transfection method.
These recombinant FzCRD proteins were used for the structural studies and the surface plasmon
resonance (SPR) binding experiments (Chang et al., 2015).

Notes:
The monoVenus protein fused to the C-terminus of FzCRD and the treatment of valproic acid
enhances the expression level of FzCRD fusion proteins in HEK293 cells. We recommend the
pHLsec-mVenus-12H vector described in Chang et al., 2015 for the protein expression and the
supplement of 4 mM valproic acid (MilliporeSigma, P4543) in a low serum medium.

Materials:
1. HEK293T (ATCC CRL-11268): used for the production of complex glycosylated FzCRD
proteins for functional studies.
2. HEK293S GnTI- (ATCC CRL-3022): used for the production of homogeneously
glycosylated FzCRD proteins for structural studies.
3. Complete medium: DMEM (MilliporeSigma, D6546) with 10% FBS (ThermoFisher
Scientific, 11573397 or 26140079), 1x L-Glutamine (ThermoFisher Scientific,
25030081), 1x Non-Essential Amino Acids (ThermoFisher Scientific, 11140050)
4. Low serum medium: DMEM (MilliporeSigma, D6546) with 2% FBS (ThermoFisher
Scientific, 11573397 or 26140079), 1x L-Glutamine (ThermoFisher Scientific,
25030081), 1x Non-Essential Amino Acids (ThermoFisher Scientific, 11140050)
5. Serum-free medium: DMEM (MilliporeSigma, D6546), 1x L-Glutamine (ThermoFisher
Scientific, 25030081), 1x Non-Essential Amino Acids (ThermoFisher Scientific,
11140050)
6. Polyethyleneimine (PEI; MilliporeSigma, 408727): prepare work solution (1 mg/ml) by
adjusting pH to 7 with HCl and filtering through a 0.22 um pore size filter to sterilize.
7. Phosphate buffered saline (PBS; ThermoFisher Scientific, 10010023)
8. Trypsin-EDTA (ThermoFisher Scientific, 25300054)
9. T175 tissue culture flasks (MilliporeSigma, C7481)
10. Plastic roller bottles (Greiner Bio-One, 681070)
11. TALON Metal Affinity Resin (TaKaRa)

Protocols:

Protein expression of FzCRD proteins in HEK293 cells

We described the protocol which was modified from a previous publication (Aricescu et al.,
2006) and use one roller bottle as an example. If lacking the equipment for roller bottles,
TripleFlask (MilliporeSigma, F8667) and HyperFlask (MilliporeSigma, CLS10030) are
alternatives.
1. Prepare a T175 flask in which HEK293 cells are about 90% confluent.
2. Remove the old media and detach the cells by rinsing with 10 ml PBS, adding 4 ml
Trypsin-EDTA, waiting for 5 min, tapping the flask gently, adding 21 ml complete
medium and resuspend by pipetting without aerating.
3. Pipette all suspension cells into one roller bottle and add 200 ml of prewarmed complete
medium.
4. Incubate at 37 oC for 3 days (HEK293T) or 5 days (HEK293S GnTI-) in a rotating
incubator. If using TripleFlask or HyperFlask, incubate at 37 oC and 5% CO₂.
5. For transfection of one roller bottle, mix 0.5 mg of DNA with 25 ml serum-free medium
thoroughly in one sterile falcon 50 ml tube. In a second one sterile falcon 50 ml tube, mix
1 ml PEI (1 mg/ml) with 25 ml serum-free medium thoroughly.
6. Combine and mix the DNA and PEI solutions, and incubate at tissue culture hood for 10
min.
7. Remove the old medium from the roller bottle.
8. Add 200 ml of prewarmed low-serum medium and add 50 ml of DNA/PEI mixture to the
roller bottle.
9. Incubate at 37°C for 5 days (up to 7 days for HEK293S GnTI-) and harvest the
conditioned media.
10. Centrifuge the conditioned media at 6000g for 20 min and filter the supernatant through
0.22 um pore size filter (e.g., Steritop Threaded Bottle Top Filter).

Protein purification of FzCRD proteins from the 0.25 L conditioned medium.
1. Use dialysis tubing (either 3.5K or 7K Dalton MWCO) for the dialysis of the conditioned
media in PBS with 0.5 M NaCl at 4°C twice.
2. Harvest the dialyzed conditioned media, adjust the Imidazole concentration to 2.5 mM,
pH 7.5, and filter through 0.22 um pore size filter.
3. Equilibrate the 0.5 ml TALON resins in Wash Buffer (25 mM Tris, pH 7.5, 0.5 M NaCl,
10% Glycerol, 10 mM Imidazole) and add into 2L conical flask containing the dialyzed
media.
4. Incubate at room temperature for 1-2 hr at 120 rpm.
5. Add a large reservoir to the top of the Econo-Column (Bio-Rad) and pour the media into
the Econo-Column to collect the TALON resins.
6. Wash the TALON resins with Wash Buffer for 100 column volumes.
7. Elute with Elute Buffer (25 mM Tris, pH 7.5, 0.5 M NaCl, 0.5 M Imidazole) completely.
8. Dialyse the collected FzCRD proteins in Dialysis Buffer (25 mM Tris, pH 7.5, 0.3 M NaCl,
10% Glycerol) overnight at 4°C and add His-tagged HRV 3C protease (home-made;
Chang et al., 2015).
9. Add 0.5 ml equilibrated TALON resins and incubate either 1-2 hrs at room temperature
or overnight at 4°C.
10. Collect the flow-through from the Econo-Column and concentrate FzCRD proteins for the
gel-filtration by using a HiLoad Superdex 200 pg column.

Reference:
Aricescu, A.R., Lu, W., and Jones, E.Y. (2006). A time- and cost-efficient system for high-level
protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62, 1243-1250.
Chang, T.H., Hsieh, F.L., Zebisch, M., Harlos, K., Elegheert, J., and Jones, E.Y. (2015). Structure
and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and
proteoglycan. Elife 4.