Expression and Purification of rat CYP27B1 1.

Incubate 5X 100 mL culture (TB broth with 50 ug/ml Amp and 25 ug/ml Kan) containing 1 mL of O/N culture (14.5 hrs, TB broth with 50 ug/ml Amp and 25 ug/ml Kan) with 225 rpm at 37C. 2. Grow the cell until OD660=0.5-0.8. It takes normally 4 hrs with 225 rpm at 37C. 3. Add 1 mM ALA and put it in the incubator with 200 rpm at 26C. 4. After 15 min, add 1 mM IPTG and 4 mg/mL arabinose and express for 21 hrs. 5. After 21 hour expression, pellet the cell with 4000 rpm for 10 min at RT and decant the supernatant. 6. Put the cell in the ice 7. Resuspend the cell with Buffer A (30 mL total) including 0.2 mM PMSF (make 1 mL of 100 mM stock dissolved in isopropanol and add 60 uL). 8. Place the tube in the shaker for 30 min to homogenize. 9. During shaking, rinse the resin (4 mL of well-mixed resin solution), which is soaked in EtOH, with deionized water by centrifuging 5 times with 3400 rpm at 4C for 2 min. Decant the supernatant and resuspend with deionized water each time. Equilibrate the resin with 2 mL of buffer A. 10. Sonicate the cell at 50% of the maximum output. Run program #8 (~30 s pulse) 6 times with 2 min interval. 11. Ultracentrifuge the cell at 4C with 13500 rpm (small rotor) for 1 hr. 12. After ultracentrifugation finished, mix the resin and the cell and put it on the shaker for 1 hr. 13. During protein adsorption, equilibrate a PD-10 column with 25 mL of Buffer A. 14. After protein adsorption, pack the column. 15. Continuously wash the column with 10 CV (20 mL) Buffer A + 50 mM imidazole. 16. Wash the column with 10 CV Buffer A + 60 mM imidazole. 17. Wash the column with 10 CV Buffer A + 70 mM imidazole. 18. Wash the column with 10 CV Buffer A + 80 mM imidazole. 19. Wash the column with 10 CV Buffer A + 90 mM imidazole. 20. Wash the column with 10 CV Buffer A + 100 mM imidazole. 21. Use the fraction collector during elution of the protein with 5 CV Buffer A + 200 mM imidazole. Note that the flow rate is reduced to 0.5 mL/min for elution. 22. Combine all collecting tubes that contain the protein and concentrate the protein with Amicon tube until obtaining 2.5 mL total. Centrifuge with 5000 g at 4C for 30 min and check the volume. 23. Add the concentrated protein of a total volume of 2.5 mL to the PD-10 column and elute with 3.5 mL Buffer A. 24. Take 50 uL of purified protein and mix it with 750 uL of buffer A to measure the absolute spectrum.

25. Prepare 1M of sodium dithionite in Buffer A and add 10 uL into 800 uL of protein solution from step 24. 26. CO bubble for 1 min and measure CO-difference spectrum. 27. Calculate the concentration of the protein and transfer it into 15 mL Amicon tube to concentrate more by centrifuging with 3350 g at 4C. 28. Check the volume after 2 hrs.

Buffer A 100 mM KPi, 0.5 M NaCl, 20% glycerol, 1% Chaps, pH 7.4 Buffer A 100 mM KPi, 0.5 M NaCl, 20% glycerol, 0.1% Chaps, pH 7.4 Absolute spectrum 1. Blank: Buffer A (700ul) 2. Sample: Add 200 ul of purified CYP27B1 CO-difference spectrum 1. Make 1M of sodium dithionite and add 10 ul to the sample solution from absolute spectrum. 2. Measure the blank. 3. CO bubble for ~ 1 min and measure the sample. The reconstitution enzyme assay Add the following order of addition to make it up to 1 ml 5 ul 25(OH)D (final concentration must be 1 uM or something) 735.2 ul Buffer A 125 ul (4.35 uM) of purified CYP27B1 9 ul ADR (55 uM) 2.8 ul ADX (350 uM) 100 ul NADPH (10 mM, 0.0017g in 200 ul) In case of using NADPH regeneration system 3 ul of G6P dehydrogenase from yeast 20 ul G6P (1 mM, 0.0076g in 500 ul)

1. Incubate in 1.5 ml eppendorf tube in ice for 5 min. 2. Place it in hot plate holders and incubate for an hour at 37C. 3. Terminate the reaction by adding 2 ml Methanol.