Growth, Purification, and Characterization of P450 cam

Transcription

1 1. Cell growth without isotopic labeling Growth, Purification, and Characterization of P450 cam Growth medium Per liter (all components are previously sterilized by either autoclave or filtration): 5 M9 salts 200 ml Sterilized distilled H 2 O 800 ml P1 metal mix 2 ml 1 M MgSO 4 2 ml 1 M CaCl ml 0.1 M FeCl ml 2% thiamine 12.5 µl Chloramphenicol (25 mg/ml) 1 ml Kanamycin (50 mg/ml) 1 ml 50% glycerol 8 ml 5 M9 salts is made by dissolving the following salts in distilled H 2 o to a final volume of 1 liter: Na 2 HPO 4 34 g KH 2 PO 4 15 g NaCl 2.5 g NH 4 Cl 4 g The salts solution is divided into 200 ml aliquot and sterilized at 121 C for 20 min. P1 metal mix is made by dissolving the following metals in distilled water to a final volume of 1 liter and filter to sterilize: H 3 BO g MnCl 2 4H 2 O 4.32g ZnCl g MoO g CuSO 4 5H 2 O 0.003g CoCl 2 6H 2 O 0.012g Growth procedure Fresh transformants: the plasmid pdnc334a (under the control of lac promoter) containing C334A mutant of CYP101 was electroporated into E. coli strain NCM533 or JM109; the transformation plate is incubated at 37 C overnight. Starter culture: inoculate a single colony from above transformation plate into 5 ml LB containing appropriate antibiotics and incubate at 37 C till OD 600 reaches 0.6 (~4 hrs). Scale up: transfer the starter culture to 50 ml fresh LB medium with antibiotics and incubate at 37 C with shaking till OD 600 reaches 0.6 (~2 hrs) Transfer to M9+ medium: spin down the cell with centrifugation at 6k rpm in a sterile centrifuge bottle, resuspend the cell pellet in 50 ml M9+ medium, and incubate at 37 C with shaking for 2 hrs. Scale up in M9+ medium: transfer the 50 ml culture in M9 into 1 liter M9+ medium, continue incubation at 37 C with shaking till OD reaches 1.0. IPTG Induction: add 70 mg δ-aminolevulinic acid to the culture and incubate for 30 min; add 1 ml IPTG (1 M) and 1 ml camphor (5 mm) to induce protein expression in the presence of camphor. Continue to 1

2 incubate at 37 C with shaking for 12 hrs. The cell color should be orange indicating well-expressed P450 cam. Storage: Spin down the cell, measure the weight of cell pellet (normally 1 liter of cell culture will resulted in ~5 g of cell paste), and store at -70 C till ready to purify. 2

3 2. Cell growth with isotopic labeling (uniformed 2 H, 13 C, 15 N-labeled) Growth medium To 1 kg of D2O (903 ml, Cambridge Isotope Lab (CIL) # DLM ), add Na 2 HPO g KH 2 PO g NaCl 0.47 g 15 NH 4 Cl (CIL # NLM-713-1) 1 g MgSO g CaCl 2 8 mg Kanamycin 45 mg (U- 13 C, 2 H) glucose (CIL # CDLM ) 3 g Filter to sterilize, and then add the following sterile additives: 0.1 M FeCl 3 90 µl Perdeuterated P1 metal mix* 1.8 ml 2% thiamine 11 µl *: made by drying the water containing P1 metal mix and re-dissolve the salts into D 2 O. Growth procedure Fresh transformants: the plasmid pet-p450 cam containing C334A mutant of CYP101 was electroporated into E. coli strain BL21(DE3); the transformation plate is incubated at 37 C overnight. Starter culture: inoculate a single colony from above transformation plate into 5 ml LB containing appropriate antibiotics and incubate at 37 C till OD 600 reaches 0.6. Scale up: transfer the starter culture to 120 ml fresh LB medium with antibiotics and incubate at 37 C with shaking till OD 600 reaches 0.6. Transfer to H 2 O-M9+ medium: spin down the cell with centrifugation at 6k rpm in a sterile centrifuge bottle, resuspend the cell pellet to 700 ml M9+ medium, incubate at 37 C with shaking till OD reaches 0.8. Transfer to D 2 O-based isotopic M9+ medium: spin down the cell with centrifugation at 6k rpm in sterile centrifuge bottles (carry-over of H 2 O solutions should be avoided as much as possible). Resuspend cell pellets in the D 2 O-based isotopic M9+ medium (~900 ml), continue incubation at 37 C with shaking till OD reaches 0.8. IPTG Induction: add 70 mg δ-aminolevulinic acid to the culture and incubate for 30 min; add g solid IPTG (final concentration: 1 mm) and 0.7 mg solid camphor (final concentration: 5 µm) to induce protein expression in the presence of camphor. Continue to incubate at 37 C with shaking for 12 hrs to 24 hrs till the culture exhibits orange color. Storage: Spin down the cell, measure the weight of cell pellet (normally 1 liter of cell culture will resulted in ~5 g of cell paste), and store at 70 C till ready to purify. Note 1: cell OD is the determinants for the transition of growth stages, while the incubation timing and temperature is flexible. 3

4 Note 2: Strain choice Choose BL21(DE3)/pET-P450 cam for uniform 13 C-labeling as (U- 13 C, D 7 ) glucose is much cheaper than (U- 13 C, 2 H) glycerol, which is a preferred carbon source for NCM533. NCM533/ pdnc334a is better for Perdeuteration of P450cam where only 15 N labeling is needed since D 8 -glycerol is cheaper even than D 7 -glucose (perdeuterated glucose, D 12 -glucose, is not available yet). For other cases, both strains are feasible. 4

5 3. Purification of P450 cam Buffers L buffer (need ~3 liters for each application): 50 mm Tris Cl (ph 7.4), 50 mm KCl, and 1 mm camphor. Vacuum degas before use. Per liter: 50 ml 1M Tris Cl (ph 7.4) 3.73 g KCl 154 mg camphor 950 ml dh 2 O H buffer (need ~ 100 ml for each application): 50 mm Tris Cl (ph 7.4), 300 mm KCl, and 1 mm camphor. Per 100 ml: 100 ml L buffer 1.86 g KCl NMR buffer (~5-10 ml): 10% D 2 O/90% H 2 O, 50 mm d11 -Tris Cl (ph 7.4), 100 mm KCl, and 2 mm camphor. Per 10 ml: 66 mg d11 -Tris Cl 1 ml D 2 O (99%) 4 ml caphor (5 mm in H 2 O) 3 ml H 2 O 74.6 mg KCl Adjust ph to 7.4 with HCl Adjust the total volume to 10 ml with H 2 O Purification procedures (Per 10 grams of cell paste) Sonication. Transfer cell paste to a small metal beaker, thaw at room temperature, and resuspend in 40 ml L-buffer. The cell suspension was then disrupted by sonication using the Misonix sonicator 8 times for 30 sec/on 30 sec/off at power setting of 5.5 (keep the sample on ice). Centrifuge at 16k rpm with the JA-20 rotor for 50 min and keep the bright orange-red supernatant. Protamine sulfate precipitation of DNA. Dissolve 200 mg of protamine sulfate in 5 ml L-buffer, add it to the protein solution dropwise with stir. Centrifuge at 16k rpm with the JA-20 rotor for 30 min and keep the supernatant. Ammonium sulfate cut. Add 490 mg ammonium sulfate ml 1 gradually to the protein solution with stirring on ice. After stirring for an additional 30 min on ice, collect the precipitant by centrifugation at 16k rpm for 30 min and dissolve the precipitant in 5 ml L-buffer. Dialysis. Add the protein solution to a dialysis bag, which was pre-treat in boiling dh 2 O for 30 min, and seal the bag with dialysis clamp. Dialyze the protein solution against 1 liter of L-buffer with stirring in the cold room overnight. Dilute the protein solution to 100 ml with L-buffer. DEAE sepharose fast-flow chromatography. Pack an anion-exchange column using DEAE sepharose fast flow resin (Amersham, catalog No ) according to the manufacturer s manual. Equilibrate the column with 200 ml L-buffer. Load the protein solution onto the column, wash the column with 100 ml L-buffer, and develop the column with a linear gradient of L-buffer and H-buffer. Collect the orange- 5

6 color fractions, measure the UV-vis spectrum of the fractions, and pool the fractions with A 390 /A 280 > 0.8 (see the next section for extinction coefficients). Concentrate the protein solution using Amicon Ultra-15 (Millipore) till the total protein volume less than ml. P-100 size exclusion chromatography. Pre-equilibrate P-100 column in the cold room with L-buffer under N 2 bubbling (takes at least an overnight run). Load the concentrated protein solution from DEAE column onto a P-100 column, elute with L-buffer with N 2 bubbling. Collect fractions with automatic fractional collector at 15 min interval. Measure the UV-vis spectrum of the colored fractions, and pool the fractions with A 390 /A 280 > 1.4. Concentrate the protein solution using Amicon Ultra-15 concentrator till the protein concentration exceeds 0.5 mm. Exchange into NMR buffer. This step can be postponed till you are ready for the NMR experiment. Add 800 µl P-2 resin (can be in any buffer) onto a spin column, spin using the microcentrifuge at 2k rpm for 15 sec; equilibrate the column with NMR buffer by applying 200 µl NMR buffer to the column and spin down for 10 times. Load the concentrated pure protein sample (the volume of the protein sample should be less than 30% of the column bed volume) onto the column, spin with the same speed, and collect the eluant. If not used immediately, should store in liquid nitrogen in aliquots. 6

7 4. Enzymatic assay Principle The activity of P450 can be measured by monitoring the rate of NADH consumption in the presence of excess of the two remaining enzymes involved in hydroxylation. Procedures To each microcentrifuge, add: 0.5 µm P450 cam 0.5 µm putidaredoxin reductase (PdR) 5 µm putidaredoxin (Pdx) Suitable amount of L-buffer to a volume to 770 µl Incubate at room temperature for 10 min. Add 330 µl NADH (1 mm in L-buffer, final concentration 330 µm), mix by inverting the tube a couple of times, and then immediately transfer to a quartz cuvette and put in the UV spectrometer. The activity can be measured by monitoring the absorbance change at 340 nm over a short period of time (normally with 1 min). 7

8 5. Protein concentration and purity. Extinction coefficient for oxidized and substrate-bound P450 cam : E 280 = 63.3 mm 1 cm 1 E 391 = mm 1 cm 1 Protein concentration (mm) = (A 391 ) 102 dilution factor Purity criteria: A 391 /A 280 =1.63 purity = 99% A 391 /A 280 =1.45 purity = 95% For our NMR purpose, A 391 /A Reference Gunsalus, I. C., and Wagner, G. C. (1978) Methods in Enzymology 52,