Production of Recombinant Annexin V from plasmid pet12a-papi

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Lynn Singleton
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1 Tait Research Laboratory Page 1 of 5 Principle Production of Recombinant Annexin V from plasmid pet12a-papi Annexin V is expressed cytoplasmically in BL21(DE3) E. coli (Novagen) with the pet vector system under control of the bacteriophage T7 promoter. Purification is by Ca 2+ dependent membrane binding and Mono Q chromatography at ph 8.0. The expected yield after concentration and final dialysis is approximately 45 mg of annexin V per liter of culture. Procedure 1. Growth of Bacteria and Protein Synthesis a. Inoculate 3, 1 liter volumes of T Broth (each liter in a 2 liter flask) containing 50 ug/ml of carbenicillin (2.5 ml/liter of a 20 mg/ml stock) with 30 ul glycerol stock of pet12a- PAP1 in BL21(DE3). b. Incubate culture for h at 37 C in a shaking incubator at 220 rpm. c. The next day, read A600 of a 1:10 dilution of the culture in broth. The A600 should be >5.0. d. Weigh empty centrifuge bottles. Divide each 1liter culture between two 500 ml centrifuge bottles (6 bottles total). Spin for 10 minutes at 5000 rpm (2,661 x g) at 4 C in a Sorvall HC5B centrifuge with a GS3 rotor e. Decant supernatant and discard. f. Resuspend the pellets in 500 ml each of cold TBS on ice. g. Spin as in step d h. Decant supernatant and discard. Weigh bottles. Determine wet weight of cell pellets. i. Store the 6 pellets at -20 C or continue. 2. Sonication and Calcium-Dependent Membrane Binding a. Resuspend each pellet in 40 ml of cold 50 mm Tris HCL, ph 7.2, 10 mm CaCl 2, on ice. b. Combine suspensions into 2 portions, each containing slurry from 3 pellets (~ 140 ml each) in 250 ml plastic beakers on ice. Sonicate each at 95% power with the large

2 Tait Research Laboratory Page 2 of 5 sonicator head for 4 x 2 min. Cool solutions to 4-10 C between 2 min pulses. Do not let samples get above 37 C or protein will denature. c. After completion of the total 8 min of sonication per solution, split each suspension into 4 x 50 ml Sepcor tubes (approx. 35 ml each). Centrifuge for 20 minutes at 17,000 rpm (22,530 x g) at 4 C in a Sorvall HC5B centrifuge with an SS34 rotor. d. Decant supernatant and discard (rannexin V is now bound to cell membranes in pellet). e. Resuspend each pellet completely in 20 ml of 50 mm Tris HCl, ph 7.2, 20 mm EDTA, on ice. (The EDTA releases rannexin V from the cell membranes in the pellet). f. Spin as in step c. Decant supernatant and SAVE. Discard pellet g. Filter supernatant with 0.45 micron filter. Store at -20 C or continue. h. Dialyze supernatant at 4 C into 20 mm Tris HCL, ph 8.0, with 3 x 2 liter changes of buffer using 6.4 ml/cm dialysis tubing. Continue to Mono Q purification step or store at -20 C (total volume is approximately 160 ml). 3. Mono Q Anion-Exchange Chromatography a. 0.2 micron filter protein solution. b. Set the A280 (1 mm) detector to 2.0 full scale c. Load sample on column (for above amount load 25 ml/run on Mono Q HR 16/10). d. Wash column until baseline is stable. e. Run gradient (0 to 30% B) and collect protein peak. f. Combine desired fractions and 0.2 micron filter. Store at -20 C or continue. Column conditions: Column Gradient Annexin V Elution position (%B) Maximum load per run (mg annexin V) HR 5/5 1.00% B/ ml HR 10/ % B/ ml HR 16/ % B/ ml

3 Tait Research Laboratory Page 3 of 5 4. Concentration and Ultrafiltration Typical concentration of rannexinv is now approximately 2 mg/ml; desired final concentration is approximately 5-8 mg/ml. 5. Dialysis Reagents a. Use Centriplus 30 (Amicon) spin filters to concentrate the samples. Maximum allowed volume is 10 ml for fixed angle rotors ie. SS-34 b. Add 10 ml of each sample to a Centriplus 30 and spin filters at 4 C at 5000 rpm (3000 x g max) in the SS-34 rotor for minutes each spin. c. Continue to discard the flow through and add more sample to the top, repeating centrifugation until desired final volume is reached such that the concentration will be around 5 mg/ml by A 280 -A 320. d. Collect retentate by spinning inverted reservoirs at 4000 rpm (2000xg) for 2 min. e. Filter (0.2µ) and save aliquot for gel and scan. Store at -20 C or continue. a. Dialyze concentrated pool into 20 mm HEPES-Na ph 7.4, 100 mm NaCl, at 4 C. b. Do a total of 4 buffer changes, 1 liter each. c. Quantitate by A 280 -A 320. (E 280 =0.6 ml/mg.cm.) d. Dilute as needed to a final concentration of 5 mg/ml with the same buffer. e. Filter through a 0.2 micron filter; aliquot and store at -70 C. f. Do QC on final aliquoted pool. 1. Plasmid pet12a-papi in E. coli BL21(DE3). Glycerol stock derived from clone #1. Date of master glycerol stock: 03/07/94 (A5CB) 2. T Broth (terrific broth): for 1 liter (sterilize in a 2 liter flask) To 800 ml Milli-Q water add: 12 g Bacto-Tryptone (DIFCO # ) 24 g Bacto-Yeast (DIFCO # ) 4 ml glycerol or 8 ml of a 50 % solution Stir until dissolved; q.s. to 900 ml and autoclave. Before use, add 100 ml of a sterile (autoclaved separately) 0.17 M KH 2 PO 4 (2.31 g), 0.72 M K 2 HPO 4 (12.54g) solution.

4 Tait Research Laboratory Page 4 of 5 3. Carbenicillin (Sigma #C-1389). Stored at -20 C. 20 mg/ml in Milli Q H2O, 0.2 micron filtered 4. 10X TBS: 50 mm Tris HCL ph 8.0, 150 mm NaCl. Store at 4 C. Dissolve 78.8 g Tris-HCL (Sigma #T-3253) and 87.7 g NaCl in 800 ml Milli-Q water. Adjust ph to 8.0 and M CaCl 2, Store 4 C 14.7 g CaCl 2.2H 2 O QS to 100 ml 0.2 micron filter mm Tris HCl, 10 mm CaCl 2, ph 7.2. Store 4 C 7.88 g Tris HCl 10 ml of 1 M CaCl 2 ph to mm Tris HCL, 20 mm EDTA, ph 7.2. Store 4 C g Tris HCL 7.60 g EDTA Na4 - (Sigma #ED4SS) ph to X A Buffer : 20 mm Tris HCL, ph 8.0. Store 4 C 31.5 g Tris HCL ph to Mono Q Buffers: Store at 4 C but warm to room temperature before use. Buffer A: 20 mm Tris HCl ph 8.0; 0.2 µ filter; degass and add 1 mm BME (Make a 1x stock from a 1:10 dilution of a 10x stock. Check ph) Buffer B: 20 mm Tris HCl ph 8.0, 1.0 M NaCl; 0.2 µ filter, degass; add 1 mm BME. Make a 1x stock directly (31.5g Tris-HCl, 58.4g NaCl ph to 8.0 and q.s. to 1 liter) Equipment 1. Shaking incubator, 37 C, for 2 liter flasks 2. Sonicator, Fisher model Vortexer

5 Tait Research Laboratory Page 5 of 5 4. HC5B Sorvall Centrifuge and GS3 and SS-34 rotors 5. Stir plate 6. Sepcor Tubes, 50 ml polycarbonate, Labcor # , VWR # Pharmacia FPLC System with Mono Q columns: HR 5/5, HR 10/10, HR 16/10 8. Filter holder for 47 mm filters, Gelman # PN Spectrophotometer 10. Freezers: -20 C and -70 C and refrigerator (4 C) Supplies 1. Millipore Millex-GS 0.22 micron filters, for small volumes, # SLGS 025 0S 2. Millipore 47 mm, 0.45 (HAWP ) & 0.2 (GSWP ) micron filters. 3. Dialysis Tubing, Spectra/Por 2, MW cutoff 12-14,000, 6.4 ml/cm VWR # Amicon Centriplus YM-30 spin filters (cat no. 4422), (30,000 MW cutoff) 5. 2 liter flasks ml centrifuge bottles, to fit Sorvall GS3 rotor cc syringes References 1. Tait, Jonathan F. and Smith, Christina. Site-specific mutagenisis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding. Arch Biochem Biophys 1991;288: Wood BL, Gibson DF, Tait JF: Increased erythrocyte phosphatidylserine exposure in sickle cell disease: flow-cytometric measurement and clinical associations. Blood 1996; 88:

OESTREICH LAB CHIP PROTOCOL

OESTREICH LAB CHIP PROTOCOL Buffers (all starred (*) buffers must be autoclaved before use) 10X Cell Lysis Buffer*: 100mL 0.214g Potassium Acetate (KOAc) = 10mM 0.147g Magnesium Acetate (MgAc) = 15mM 10mL