Recombinant Catalytic Subunit Preparation
(cAMP-dependent Protein Kinase A)

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


Clone Culture, Expression & Harvest

1. Prepare 12 liters of sterilized YT Media by placing 18 YT 
	capsules/Liter of MQ H20 and Autoclaving.  Let the media cool 
	to at least 37oC and add to each flask 1 ml of a 100 mg/ml MQ 
	H20 filtered ampicillin solution (1.2 g Ampicillin/12 ml H2O).   

2. Add 1.1 ml of sterile YT to the frozen bacterial cell stock.  
	Vortex well. Using sterile techniques, add 100 ml of this 
	solution to each flask. If you inoculate the media that is at 
	room temperature, let the cells grow for 8-8.5 hours 
	at 37oC 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.

**Alternate Method:  Inoculate cold media flasks (with 
	Ampicillin) at night. Place flasks in the shaker and set the 
	temperature to 37oC and RPM to 225. Set timer to start in the 
	early morning, ex: 1:00 or 2:00 am. Let grow 8-8.5 hours at 
	37oC until OD600 is approximately between 0.55-0.80. Record the 
	OD in your notes.

3. When cultures reach an OD600 of 0.55 to 0.80, induce them with 
	0.5 M IPTG (1.62 g/12 ml). Add 1 ml to each liter observing 
	sterile technique. Final concentration of IPTG ==> 0.5 mM.  
	Adjust the temperature control of the shaker 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.  

A Day Before Your Prep Starts or Early the First Day of Your Prep:

6. Prepare P-11 phosphocellulose resin
	A. Stir 250 mls of 0.5 M NaOH into 12-13 g of P-11 and let sit 
		for 5 minutes
	B. Filter off the supernatant and wash with MQ water (about 2 
		liters) until the pH is below 11 (Use pH paper to measure 
		approximate pH's)
	C. In filter funnel mix resin with 250 mls 0.5 M HCl and let 
		sit for 5 minutes
	D. Filter off the supernatant and wash with MQ water (about 2 
		liters) until the pH is above 3.0
	E. Transfer the P-11 into approximately 3 volumes (300 mls) of 
		10X P-11 running buffer
	F. Titrate the slurry with NaOH until the pH is 6.5. Let the 
		solution sit in the cold room
	G. Decant the supernatant and add 250 ml of 1X running buffer.  
		Stir the slurry and pH again to 6.5
	H. Leave in cold room overnight or until you add the total cell 
		supernatant.

Day 1 of Cat Protein Prep

1. Remove frozen pellets from -70oC freezer and let thaw at room 
	temperature on bench or in a beaker of water.  Resuspend the 
	thawed pellets in 240 mls 1X P-11 lysis buffer [Dilute 
	10X P-11 (cold room) and add 43 ml b-me to 5mM]

2. Lyse the cells in the French Press 2X. Save 20 ml of the total 
	cell lysate and record the total volume

3. Spin the lysate for 40 minutes at 15,000 RPM (JA-20 rotor) in 
	50 ml centrifuge tubes. Collect and combine the supernatants.  
	Take a 20 ml aliquot and record total volume. Save the pellet 
	from one of the centrifuge tubes and resuspend with water 	in 
	the same volume as total cell lysate. Take a 20 ml aliquot and 
	save.  

4. Measure and record the initial conductivity of the supernatant. 
	Dilute the supernatant with cold water (4oC) until the  	
	conductivity reaches 1.2 mS/cm. Check the pH and make sure it 
	is at 6.5.  Save a 50 ml aliquot of the diluted supernatant and 
	record volume.

5. Check pH of the resin (6.5). Decant off supernatant of resin.  
	Add resin to the diluted cell supernatant. Mix Slowly (Batch 
	bind) for 3 hours minimum (or overnight) in an appropriately 
	sized beaker.

End of Day 1 or Day 2 of Cat Protein Prep

6. Decant resin through funnel (plastic). Resuspend in 1X Running 
	buffer.

7. Pour Slurry into a 100 ml column. Pack and wash with 200 mls 
	of 1X running buffer (or batch wash the resin in a 
	beaker for 10 minutes at 4oC.).

8. Column wash with 200 mls of: 1X Running buffer + 50 mM KPO4.  
	Collect elution in bottle or beaker and save. (or batch 
	wash in beaker for 10 minutes).

9. Wash with 200 mls of:  1X Running buffer + 90 mM KPO4. Collect 
	elution in bottle or beaker and save. (or batch wash in 
	beaker for 10 minutes and pour column).

10. Elute with 250 mls of 1X Running buffer + 250 mM KPO4.  
	Collect 8-10 ml fractions.  

11. Run 12.5% SDS PAGE gels. Include standard, total cell lysate 
	(2 ml), lysate pellet (2 ml), lysate supernatant (2 ml), 
	diluted lysate supernatant (5 ml), flow through (5 ml), 1X 
	Running Buffer Wash, (20 ml), 50 mM Wash (20 ml), 90mM Wash 
	(20 ml), and selected fractions (20 ml).

12. Pool wanted protein (proteins that show little or no 
	contaminating bands) in a small beaker. Dialyze the 
	protein in 2L of Buffer A (20 mM KPO4), pH 6.5 overnight.

Day 2 or 3 of Cat Protein Prep

1. Check pH of Dialysis buffer, make sure it is at pH 6.5.

2. Measure the concentration of your dialyzed protein.  Record 
	concentration and volume of your pooled protein.  Take a 20 ml 
	aliquot.

3. Load up to 50-60 mgs of protein onto Mono S 10/10 HR of FPLC.

4. Watch for peaks. Protein usually starts eluting between 15-25% 
	of Buffer B.

5. Run a 12.5% SDS PAGE Gel.  Include standard, load (10 ml), and 
	all necessary fractions

6. Run IEF Gel to determine peaks of individual fractions (if 
	necessary).

7. Check Cat activity and record.

8. Dialyze protein for crystallographers in :
		50 mM Bicine
		150 mM Ammonium Acetate
		10 mM b-me         
		pH 8.0

9. Dialyze protein to be frozen at -20oC in:
		100 mM MOPS
		150 mM KCl
		0.1 mM DTT           
		pH 7.1

10. Record total amount of protein recovered, separating them by 
	peaks.

11. Record distribution of protein and amount distributed.


Buffers for Recombinant Cat Preparation


Lysis buffer: 	1X			10X
		30 mM MES		300 mM MES
		1 mM EDTA		10 mM EDTA
		50 mM KCl		500 mM KCl
		5 mM b-me		
		pH 6.5			pH 6.5

P-11 Running buffer:	
			1X			10X
			30 mM MES		300 mM MES
			1 mM EDTA		10 mM EDTA
			5 mM b-me		
			pH 6.5	 		pH 6.5

Mono S:
			Buffer A:		Buffer B:
			20 mM KPO4 		20 mM KPO4
						1M KCl
			5 mM b-me or		5 mM b-me or
			  2 mM DTT		  2 mM DTT
			pH 6.5	     		pH 6.5

 S-100:			1X:			10X:
			200 mM KCl		2 M KCl
			25 mM KPO4		250 mM KPO4
			5 mM b-ME
			pH 7.0			pH 7.0

For all solutions: pH when cold, add b-me to final solutions only 
(350 ul/1L).