HIV-1 NC Prep 6/12/2001 JLN Adapted from RD 1998. Preliminary note: We noted that the main factor in increasing expression levels for HIV-1 NC from the pRD2 plasmid was transforming the cells and plating them onto ZB plates. Plating the transformaitons onto LB plates resulted in very low levels of expression. Also, induction at 0.5 OD600 resulted in the best expression levels, although each colony should be tested separately for expression levels. It was also seen that stopping growth after 4.5 hrs. resulted in highest levels of protein expression. It is also important to use BL21pLysE. When purifying NC, concentrating more than 1L after the ion exchange step results in precipitation and lower yield. If purifying more than 1L, run 1 batch/L on gel filtration column. In summary, keys to high expression: 1. Plate transformation onto ZB plates. 2. Induce at 0.5 OD 600 units. 3. Grow cells for 4.5 hours, then harvest. Part I: Tranformation Part II: Expression test Part III: Large scale cell growth Part IV: Purification Part I: Tranformation Make ZB Amp/Chloramp plates for transformation. DO NOT MAKE LB Amp/Chlormp PLATES!!!!! ZB plate recipe: 100 mL Tryptone 1 g NaCl 0.5 g Agar 1.5 g After autoclaving, add 100 uL of 1000x Chloramp and Amp stocks. Make sure solution is not too hot, or will kill antibiotics. Chloramp stock: 34 g/L, make up in EtOH Amp Stock: 100 g/L Part I: Tranformation Perform transformation as follows: 1. Autoclave ZB for ZB Amp/Chloramp plates. 2. Label eppendorf tube for each rxn., ice 5 min. 3. Get BL21 pLysE cells from freezer, keep on ice. 4. Thaw DNA at RT 30 min. 5. Heat Block low setting (8), ~42C. 6. Get SOC media from freezer, thaw at RT. 7. Add 50 uL cells, 2uL plasmid to each labeled and iced eppendorf. Ice 30 min, heat shock 40s, ice 2 min. 8. Add 100 uL SOC to each tube from 7. 9. Incubate 1hr (250 rpm, 37C), make sure tubes are oriented horizontally. 10. Plate 10, 20, 30 uL cells onto ZB Amp/Chloramp plate. Place in 37C room overnight. Do not let colonies overgrow. Part II: Expresson test ***** 5 mL expression test ***** Pick three colonies, innoculate 5 mL LB Amp/Chloramp For each innoculated colony, streak fresh ZB Amp/Chloramp plate Put plates in 37C overnight Grow cells to OD ~0.5, induce w/ 1M IPTG. Take 300 uL samples for expresion test. Add 20 uL dye/OD unit. Take samples every hour, up to 5-6 hours.... expression max ~ 4.5 hrs. Check expression with SDS gel, load 12 uL. Part III: Large Scale growth ***** Large scale growth 6 x 4L ***** Use 4L notched flasks for better aeration Prepare 6 x 1L LB Amp/Chloramp media Set up 6 x 5mL LB Amp/Chloramp starter cultures overnight. Spin down cells @ 3000g in morning, transfer to 100 mL LB Amp/Chloramp Grow 100 mL cultures to OD600 = 1.0, spin down cells @ 3000g, resuspend in 1L. Grow cells to OD600 = 0.5, induce with 1M IPTG. Harvest cells 4-5 hours after induction. Take 1 mL samples, add 30uL dye/OD unit. Load 10 uL onto SDS gel. Part IV: Purification Buffers and stock solutions: Lysis Buffer 1L 50 mM Tris-HCl pH 8.0 7.88g 10% Glycerol 100mL 100 mM NaCl 5.844g 100 uM ZnCl2 * 500 uL 0.2M stock 5mM DTT * 0.771g 2mM EDTA * 4 mL 0.5M stock * = Add after autoclaving SP-A Buffer 1L 50 mM Tris-HCl pH 8.0 7.88g 10% Glycerol 100mL 100 mM NaCl 5.844g 100uM ZnCl2 * 500 uL 0.2M stock 5 mM BME * 352 uL (14.2M stock) * = Add after autoclaving SP-B Buffer 500 mL 50 mM Tris-HCl pH 8.0 3.94g 10% Glycerol 50 mL 1M NaCl 29.22g 100 uM ZnCl2 * 500 uL 0.2 M stock 5 mM BME * 176 uL (14.2M stock) * = Add after autoclaving NMR Buffer: 200 mL 25 mM d-NaOAc pH 6.5 0.4253g 25 mM NaCl 0.2922g 100 uM ZnCl2 * 100uL 0.2M stock 100 uM BME * 1.4 uL * = Add after autoclaving Note: if NOT making NC for NMR, can use NMR buffer for Gel Filtration column and exchange buffer Pepstatin A 1 mg/mL 1g in 10 mL EtOH PMSF 10 mM stock in EtOH 0.0871g in 50 mL PEI 10% stock pH 7.9 Dilute in lysis buffer for 4% solution Deoxycholate 0.1% solution 100 mg in 10mL lysis buffer At this point, autoclave glass tubes for the FPLC. Will process 2L for purification. 1. Thaw cells 30 min in ice water. 2. Add 172 uL 10 mM PMSF, 30 uL 1 mg/mL Pepstatin A. 3. Add 2.1 mL 1% deoxycholate. 4. Keep on ice 20 min. 5. French press 2x. 6. Add 4.5 mL 4% PEI to precipitate nucleic acids, stir on ice 45 min. NOTE: make sure pH of PEI is 8.0!!! 7. Transfer Solution to two cold 50 mL centrifuge tubes (autoclaved). 8. Pellet @ 37,000 rpm (33,000g) for 30 min at 4C. 9. Filter Supernatant thru 0.22 uM syringe filter, collect in cold 50 mL Falcon tube. FPLC purification. All programs are stored on the AKTA computer. Briefly, two columns are run in series. Solution first flows thru Q column, then thru SP column. Can purify 3L in one run. Three different programs to run: NC Load NC Elute NS Superdex 75 Load program Flow rate: 3 mL/min Wash column w/ 4 CV buffer A. Reequilibrate with 2 CV buffer A. Elution program Flow rate: 5 mL/min Wash column with 2 CV Start frac at 40 %B Elute frac size: 2.5 mL Peak start slope: 100 mAU/min Peak end slope: 75 mAU/min Minimum peak width: 2.00 min End Frac at: 100 %B Target concentration B1 40 %B Length gradient 1: 2 Target concentration B2 60 %B Length gradient 2: 6 Target concentration 3: 100 %B Clean with 1 CV Reequilibrate with 1.5 CV Concentrate eluent to <5mL in centriprep 3 NC Superdex 75 Filter sterilize concentrate from elution thru spartan 0.45 uM filter. Isocratic elution Flow Rate: 0.25 mL/min (?) Use non-deutreated NaOAc buffer if not using for NMR studies. Typical yield: ~25 mg/L