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 37íC 
	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 37íC 
	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 37oC 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 37oC 
	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 24oC.  Leave lid open 
	or the temperature will continue to rise past 24oC.  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 -70oC 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).