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Recombinant His-6-Cat Purification
Recombinant His-6-Cat Purification
(cAMP-dependent Protein Kinase A)

Susan Taylor (University of California at San Diego) January 3, 1997

 	---Typical yield approximately 15 mg per liter of cells 	---Use this protocol for most His-6-Proteins including GFP

Growing Cells and Expressing Protein

 1. Prepare 6 liters of YT Media by placing 18 YT capsules/1 Liter  	MQ H2O and autoclaving.  Let the media cool to at least 37&deq;C  	and add to each flask 1 ml of filtered 100 mg/ml Ampicillin  	stock. (0.6g Ampicillin/6ml H2O)    2. Add 0.5 ml of sterile YT to the frozen bacterial stock.  Vortex  	well.  Using sterile technique, add 100 ml of this solution to  	each flask.  If you inoculate the media that is at room  	temperature, let the cells grow 5-7 hours in the shaker at 37°C  	until OD600 is greater than 0.55 and less than 0.80.  If you  	inoculate media that is warm, the cells will grow to the  	appropriate OD in less time than predicted.  ***Alternate Method:  Inoculate media that is at room temperature  	after addition of sterile Ampicillin in the evening. Place  	flasks in the shaker and set the temperature to 37°C and RPM to  	225. Set the timer to start the shaker early in the morning,  	ex: 3:00 or 4:00 a.m. Let the cells grow 5.5-7 hours at 37°C  	until OD600 is between 0.55-0.80. Record the OD in your notes.  3. When cultures reach an OD600 of 0.55-0.80, induce them with  	1.0 M IPTG (1.62g/6ml). Add 1 ml to each liter observing  	sterile technique.  Final concentration of IPTG in the media  	==> 1.0 mM.  	Adjust the temperature to 24°C.  Leave lid open  	or the temperature will continue to rise past 24°C.  Grow for  	an additional 6 hours.  4. Harvest the cells by spinning in 1 liter bottles for 11 minutes  	at 5,000 RPM. Use Sorvall centrifuge.  5. Consolidate the pellets in a 40 ml centrifuge tube. Label  	completely and store the cells in the -70°C freezer.   

Isolation of Protein from Bacteria (Day 1)

 1. Resuspend cell pellet in 60 mls of His6-Cat buffer [Dilute 10X  	His6-Cat buffer and add 22 ml b-me, pH 8.0]  ***If the resuspended cells are viscous, sonicate several times  	with large probe then french press  2. Lyse the cells with two passes through a french press.  Save 20  	ml aliquot of the total cell lysate and record the total  	volume.    3. Spin the lysate for 40 minutes at 15,000 RPM in 50 ml  	centrifuge tubes.    4. While the lysate is spinning, equilibrate the Ni2+ resin. Use  	15 mls of resin for a 6 L WT His6-Cat prep, less for mutants.    	Equilibrate by putting the resin in a 50 ml conical tube and  	spinning down the slurry. Decant the supernatant and resuspend  	the packed resin in His6-Cat Buffer, pH 8.0. Repeat several  	times.  Load the resin onto a column or keep in conical tube  	for batch method purification.  5. When lysate spin is complete, collect and combine the  	supernatants.  Take a 20 ml aliquot of the supernatant and  	record the total volume of supernatant pooled. Save the pellet  	from one of the centrifuge tubes and resuspend with water in  	the same volume as the total cell lysate. Take a 20 ml aliquot  	of the resuspended pellet and save.  6. Check the pH of the supernatant protein solution.  Re-pH, if  	necessary to 8.0. Load protein supernatant onto the column  	slowly (1 ml/min). Collect the flow through and pass over  	column again. Wash with 5 column volumes His6-Cat lysis buffer.  ***Alternatively, batch bind the supernatant with the Ni2+ Resin  	for at least 30 minutes (longer if necessary...but not more  	than 2 hours). Then, spin the resin down into one 50 ml  	conical tube. Wash the resin in the conical tube 2 times with  	30-40 mls of H6-Cat Lysis Buffer.  7. Step elute His-6-Cat from Ni2+ (either batch method or on  	column) with:    	1) 2 column volumes (Ex: 30 + 30 mls) His6-Cat lysis buffer +  		20 mM imidazole, pH 7.0   	2) 2 column volumes Cat lysis buffer + 50 mM imidazole, pH 7.0   8. Elute the protein off the resin with 10 column volumes (150  	mls) Cat lysis buffer + 100 mM imidazole, pH 7.0. Collect  	small fraction sizes, ex: 5 ml fractions.    9. Run 12.5% SDS PAGE gels. Include standard, total cell lysate  	(2ml), lysate pellet (2ml), lysate supernatant (2ml), flow  	through (5ml), Wash #1 (20ml), Wash #2 (20ml), 20 mM Wash  	(20ml), 50 mM Wash (20ml), and selected fractions (20ml). 

Cleaning up the Protein (Day 2/3)

 1. Pool wanted proteins in a small beaker or conical tube.   	Dialyze the protein in 2 L of Buffer A (20 mM KPO4).