GP44/62 Clamp Loader Complex

The clamp loader serves to load the processivity clamp onto duplex DNA. It is composed of four subunits of GP44 and one subunit of GP62. It can also act as a chaperon for recruiting GP43 to assemble the functional holoenzyme (GP43, GP45, GP44/62). This was the biggest pain of a protein to purify. We have many constructs of this protein with many different purification schemes and cells lines. If you would like to the details, please consult Greg Hild’s lab notebook.

Contents

1. Transform
2. Express
3. Purification
   1. DEAE Column
   2. Hydroxyapatite
   3. SP Column
4. Storage

1. Transform

1. Transform 1 uL of GP44/62 plasmid (pLY1244) into 50 uL of BL21(DE3) cells.
2. Flick 5 times.
3. Place on ice for 5 minutes.
4. Place in a 42C water bath for 42 seconds.
5. Place on ice for 2 minutes.
6. Inject the cells into 500 uL of SOC and place in a shaking incubator for 1 hour at 37C, 250 rpm.
7. Plate 250 uL of cells onto an LB-Carb plate and place in a 37C incubator for 24 hours.
8. You can hold plate for 2 weeks in 4C if you parafilm the edges.

2. Express

1. Take one cell from BL21(DE3) plate and inoculate 100 mL of LB in a 500 mL flask with a 50 ug/mL concentration of carbenicillin.
2. Grow the flask overnight for 12-18 hours.
3. In the morning, inoculate 6L of LB with 15 mL of overnight per liter.
   • Each liter should have a carbenicillin concentration of 50 ug/mL.
4. Grow until an O.D. 600 of 0.6-1.0.
5. Once the O.D. 600 is reached, inoculate the cultures with a concentration of 10 mM IPTG (2g/L).
6. Allow to grow for 1 hour and then harvest.
7. Harvest by centrifuging for 10 minutes at 5000 rpm.
8. Decant the supernatant and keep the pellet. Do this in the cold room
9. Resuspend the pellet to 70 mL with resuspension buffer.
   • 50 mM Tris pH 7.5
   • 200 mM NaCl
   • 10% Sucrose
   • 1X HALT EDTA free protease inhibitor
10. Flash freeze cells in liquid nitrogen and store in -80C until ready to use.

3. Purification

1. Either continuing from unfrozen lysate or thawing the lysate on ice.
2. The lysate was clarified via sonication and then ultra-centrifuged for 35 minutes 35000g at 4C.

3.1 DEAE Column

1. The lysate was dialyzed overnight in buffer 44HA.
   • Buffer HA:
     • 20 mM Potassium phosphate pH 7.0
     • 10 mM BME
     • 5 mM MgSO4
     • 10% Glycerol
2. A 20 mL DEAE column was equilibrated with Buffer HA.
3. The clarified lysate was loaded onto the DEAE column (the protein does not bind under these conditions).

3.2 Hydroxyapatite

1. The flowthrough from the DEAE column was collected and loaded onto a 5 mL hydroxyapatite column equilibrated with buffer HA.
2. The protein was eluted from the hydroxyapatite column with Buffer HA + 0.4 M PO4- pH 7.0.
3. Fractions were collected.
4. The fractions were analyzed via 10% SDS-PAGE.
5. Fractions containing protein were dialyzed into Buffer HA overnight.

3.3 SP Column

1. Dialyzed protein was loaded onto a 1 mL SP column.
2. The SP column was washed with 10 Column volumes of HA Buffer.
3. The SP column was eluted with a gradient using HA buffer and HA + 0.4 M PO4- pH 7.0.
4. The fractions were collected and analyzed via 10% SDS-PAGE.

4. Storage

1. Fractions with protein were combined and dialyzed into storage buffer.
   • Storage buffer
     • 20 mM Tris pH 7.0
     • 100 mM NaCl
     • 10 % glycerol
2. After dialysis, the fractions were aliquoted into 500 uL eppendorf tubes (10-20uL per tube).
3. These tubes were flash frozen in liquid nitrogen and stored at -80C.

Note: (from Scott Nelson) gp44/62 is fairly stable and can be re-frozen upon use; however, make sure it is always on ice when taken out of the -80C