TITLE: Expression and Purification of TailSpike 1 (TSP1) Protein from E. coli
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocoll adds greater clarity and stepwise-discriptions regarding the purification of tailspike protein 1 (TSP1). The structure of TSP1 is 4OJ5. </div></div>

[STEPS]
?. [Prepare LB Broth and Grow Overnight Culture]
Prepare 5-6L of LB broth in 4 liter baffled flasks. Sterilize for 20min. Cool in 37C with shaking From a plate, inoculate a single colony into 300mL LB broth with 200ul carbenicillin (50mg/ml). Grow Overnight.
Cooling LB broth in shaker at 37C with minimal shaking reduces lag time when inoculating cultures the following morning.
?. [Inoculate, Grow and Induce ]
Inoculate the 4L, baffled flasks containing LB broth with 20-50mL of the overnight culture. Adjust shaking speed to 180 rpmInoculate in the afternoon such that OD600 is between 0.8-1.0 before you leave. ABout 30 min before, cool incubator down to room temperature (21-25C)Induce with 0.10% arabinose (10g in 1L ).Grow overnight at RT until 9a or 10a the following morning.
Do not add antibiotics to the broth in the flasks.A 4-hr induction may also be done with 0.25% arabinose, but the yield will not be as high (about 1-2mg/L). Overnight induction yield at 0.10% will be between 4-8mg/L.
?. [Purify TSP1]
Spin cultures down in 1L bottles between 4500-5000xg for 20 min using Beckman Coulter rotor JLA 8.1 or 9.1. Resuspend culture in 1x PBS pH 7.4 with 10-20mM imidazole (MW 68.1) in 25-32mL. Transfer to 50mL conical tube.Place conical tube in 60C water bath for 15 min. Cool to RT.Add 5-10mg lysoszyme. Add 20ul Benzonase. Invert to mix. Lyse cells using french press or other method e.g. sonicationTransfer lysate into 30mL round-bottom centrifuge tubes and spin at 20,000xg for 20-30min. Decant into new tubes.Add 1mL resuspended Ni-NTA resin for each liter of induced culture to lysate.Incubate with end/end rotation for 30 min.Pellet resin by centrifuging for 5 min at 4000-5000xg. Carefully decant. Do not allow resin to be decanted.Combine resin into single 50mL conical tube. Wash with 1x PBS and 20-40mM imidazole for 4-5, 50 mL column volumes.After final wash, combine all resin into 15mL conical tube. Wash 1 CV. Centrifuge.Carefully remove supernatant with electronic pipetter and serological pipette.Add 2mL elution buffer (PBS 300-500mM imidazole). Incubate with end/end rotation for 5-20 min.Centrifuge. Collect supernatant in new tube, careful not to disturb resin.Repeat 3 times, each with 2mL.Quantify protein concentration using spectrophotometric or other technique.
After resuspension in PBS and transfer to 50mL conical tubes, the samples may be placed in -20C freezer overnight. Do not add benzoase or lysozyme before freezing.TSP1 is a stable trimer and steps may be carried out at room temperature.Incubating resuspended pellets at 60C for 15 min helps lyse cells and deactivate proteases.
?. [Dialyze TSP1 and Purify on Mono Q Column]
In 5 L container, add 20mM Tris pH10 (MW:121.1) and 20mM NaCl (MW:58.4).Filter protein through 0.2uM filter.Dialyze overnight using casettes or dialysis bag.The following morning, concentrate in 100,000 MWCO protein concentrator to about 500 ul total volume.From the dialysis buffer, pour 1 L into separate container. Adjust to 300mM NaCl. Degas.Perform pump wash on pump B with 300mM solution. This will be the elution buffer on mono Q column (anion exchange column).Perform protein loading, washing and purification according to manufacturer's protocol.TSP1 should elute from mono Q column in single peak.Use protein concentrators to concentrate eluted TSP1 to desired concentration.Dialyze overnight in 1x PBS supplemented with 10% glycerol.Adjust concentration to 3-4mg/ml. Use liquid nitrogen to snap-freeze tubes in 200ul aliquots.Store tubes containing TSP1 protein in -80C freezer.
I add Tris powder to 5L container (121.14*5*0.02 = 12.11g) and not buffered Tris solution. The unbuffered Tris is usually around pH10. It's not an ideal buffer in this range but that's okay. pH needs to be about 2 pH units higher than TSP1 isoelectric point around 7.