Procedure for protein expression and purification
E. coli BL21 rosetta cells transformed with a plasmid containing the clone of interest and pET15-MHL were grown in 1.0 liter Terrific Broth (Merck) medium at 37 °C until reaching OD600 of approximately 0.8. Protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C for 16h. Cells were collected at 6500 rpm in JLA 8.1000 rotor (Beckman) for 20 min and re-suspended in lysis buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole) supplemented with 1 mM tris(2-carboxyethyl) phosphine (TCEP), and 1:1000 (v/v) Protease Inhibitor Cocktail set III (Carbiochem). Cells were lysed by sonication, and clarified lysates were obtained by centrifugation at 15,000 x g in JA 16.250 (Beckman) for 60 min. Lysates were loaded onto Ni-NTA column (GE Healthcare), washed with 50 mL washing buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 1mM TCEP) and gradient eluted from 60-300 mM Imidazole. All fractions were collected and loaded in SDS-polyacrylamide gel electrophoresis (Bio-Rad). The eluted proteins were treated overnight with TEV (Tobacco Etch Virus) protease at 4 °C to remove the His6-tag. Selected fractions were pooled together and concentrated for further purification by size-exclusion chromatograph using a HiLoad Superdex S75 16/60 column (GE Healthcare) buffered in 10 mM HEPES pH 7.5, 500 mL NaCl, 5% glycerol and 1 mM TCEP. Purified proteins were finally concentrated on Amicon® Ultra (Millipore) employing 10 kDa cut-offs. The intact mass of the proteins was confirmed by electrospray ionization/time-of-flight mass spectrometry (Agilent Technologies).
The solutions were prepared according to the instructions of the M9 SeMET High-Yield growth media kit package (MD045004-50L, Orion enterprise inc, Northbrook, IL, USA). Amendments (final amounts) per liter, added in a sterile manner to 900 - 950 ml autoclaved water:
"A": MD045004A (Na/K/P/C/N sources + non-inhibitory amino acid cocktail: EDRHAPGSQNW), one pouch for 1 Liter cell culture, added just before growth: 6 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 4.4 g glucose, non-inhibitory amino acids 200 mg each (1-glutamate, 1-aspartate, 1-arginine, 1-histidine, 1-alanine, 1-proline, 1-glycine, 1-serine, 1-glutamine, 1-asparagine, 1-tryptophane)
"B": MD045004B (Mineral supplements), stored at 4°C, light avoided. 10 ml for 1 Liter cell culture: 5 mg EDTA, 430 mg MgCl*6 H2O, 5 mg MnSO4, 10 mg NaCl, 1 mg FeSO4*7H2O, 1 mg Co(NO3)2*6H2O, 11 mg CaCl2, 1 mg ZnSO4*7H2O, 0.1 mg CuSO4*5H2O, 0.1mg AlK(SO4)2, 0.1mg H3BO3, 0.1 mg Na2MoO4*2H2O, 0.01 mg Na2SeO3, 0.1 mg Na2WO4*2H2O, 0.2 mg NiCl2*6H2O.
"C": MD045004C (Vitamins), added to 50 ml of water and mixed, stored at 4°C for short term, at -20°C for long time use, light avoided after dissolution. 1 ml for 1 Liter cell culture: 1 ug thiamine (vitamin B1), 2.7 ug vitamin B12.
"D": MD045004D (inhibitory amino acid cocktail: VILKTF + SeMet). One pouch dissolved in 250 ml. 20 ml for 1 Liter cell culture: 25 mg each of 1-valine, 1-isoleucine, 1-leucine, 1-lysine, 1-threonine, 1-phenylalanine and 15 mg of selenomethionine.
"E": For MD045004E (IPTG). One pouch dissolved in 50 ml of water. 1 ml for 1 Liter cell culture. Final concentration: 1 mM isopropylthio-beta;-d-galactoside (IPTG).
Selenomethionyl proteins were produced in BL21-(D3) - a strain not auxotrophic for methionine- using feedback inhibition of methionine biosynthesis. On day 1, one colony of the transformed E. coli BL21-(DE3)-V2R-pRARE2 was transferred to 5 mL LB medium supplemented with carbenicillin (100 µg/mL) and chloroamphenicol (34 µg/mL) in a 10 ml tube and grown at 37 °C, 220 rpm overnight. On day 2, 100 ml of freshly prepared "A" solution, supplemented with "B", "C", glycerol and antibiotics were inoculated with 0.5 ml of the LB culture and grown overnight in a 200 ml flask at 37°C, 220 rpm. On day 3, 1.8 L/ 2 L bottle of freshly prepared "A" solution, supplemented with "B", "C", glycerol, antibiotics and 0.5 ml antifoam A204 (Sigma) were inoculated with 20 ml of the flask cultures and grown in the LEX system at 37°C for ~ 4 h until OD600 ≈ 1.2. Then "D" was added, immediately the cultures were cooled down to 20°C (the cooler was set to 18°C). After 15 min IPTG was added and induced overnight. In the morning of day 4, the OD was taken and the cells harvested.
The cleared lysate was loaded onto a 1.0-2.5 mL Ni-NTA (Qiagen) column (pre-equilibrated with Binding Buffer) at approximately 1.5-2.0 mL/min. The Ni-NTA column was then washed with 150 mL of Wash Buffer at 2-2.5 mL/min. After washing, the protein was eluted with Elution Buffer. The eluted sample was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gelfiltration Buffer. The fractions corresponding to the eluted protein peak were collected and further concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel. The concentrated protein (23.37 mg/ml) was stored at 4°C. For long term storage, the protein was flash frozen and stored at -80°C.
The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 30 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 degC were thawed overnight at 4 °C on the day before purification. Prior to sonication, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. After 6 minutes sonication, the cell lysate was centrifuged using a Beckman JLA-16.250 rotor at 16000 rpm for 1 h at 4 degC.
Concentration: 23.37 mg/ml
Alpha-chymotrypsin (1 mg/ml) was added to protein at 1:100 (V:V) ratio prior to crystallization, final concentration: 10 µg/ml.
Crystal condition: 20% glycerol plus 8% PEG8000, 0.1M Tris-HCl, pH8.5, sitting drop vapor diffusion, 293K.