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Expression and Purification of the CPC

 

Materials Needed:

Constructs: 6-His Aurora B/ INCENP bicistronic vector in pET28

                   Untagged Survivin/Dasra A bicistronic vector in pET15

Transform into BL21-pLysS E.coli.

 

2XYT (for 1L, scale appropriately)

900 mL ddH2O (Milli-Q)

16 g Bactotryptone

10 g Bacto yeast

5 g NaCl

 

8X Binding Buffer, pH 7.9:

40 mM Imidazole

4 M NaCl

160 mM Tris-HCl, pH 7.9

 

8X Wash Buffer, pH 7.9

240 mM Imidazole

4 M NaCl

160 mM Tris-HCl, pH 7.9

 

4X Elution Buffer, pH 7.9

1.2 M Imidazole

2 M NaCl

80 mM Tris-HCl, pH 7.9

 

Lysozyme (Sigma)

PMSF, 100 mM stock in Isopropanol 

LPC, 1000X mix (leupeptin, pepstatin, chymostatin, 10 mg/mL stock in DMSO)

 

Ni-NTA beads (Qiagen)

 

SLA-3000 rotor (Sorvall)

SLA-600 rotor (Sorvall)

SS-34 rotor (Sorvall)

 

Flasks, Graduated Cylinders, centrifuge bottles, 50mL conical tubes, eppendorf tubes (both 1.5 mL and 2.0 mL).

 


 

 

The night before:

Inoculate 100 mL 2XYT cultures with 1 colony (or glycerol stock scraping) of appropriate construct.  Select the Aurora B/INCENP culture with kanamycin, and the Survivin/Dasra A culture with ampicillin.  Grow to saturation at 37C overnight.

 

The next morning:

For Survivin/Dasra A:  Inoculate one 1L 2XYT culture with 20 mL of the Survivin/Dasra A culture plus ampicillin.  Grow cultures at 37C, shaking at 275 rpm to an OD600 between 0.6 and 0.8.  The Survivin/Dasra A culture will reach this OD in approximately 2 hours.  Take a 1 mL sample, centrifuge at top speed in a minicentrifuge, and resuspend in 100 μL of SDS-PAGE sample buffer for gel analysis.  Cool culture to room temperature.  Induce expression with IPTG to a final concentration of 1mM.  Express protein at room temperature for 4-5 hours.  Take a 1 mL sample, centrifuge at top speed in a minicentrifuge, and resuspend in 100 μL of SDS-PAGE sample buffer for gel analysis.  Harvest bacteria by centrifugation, (5000 rpm in SLA-3000 rotor, 4C, 15 minutes) wash pellet with PBS, transfer to a 50 mL conical tube, pellet again (5000 rpm, SLA-600 rotor, 4C, 15 minutes) and then freeze at -80C.

 

For Aurora B/INCENP:  Inoculate two 1L 2XYT cultures with 20-25 mL each of the overnight Aurora B/INCENP starter culture plus kanamycin.  The Aurora B/ INCENP construct tends to grow more slowly, and generally take 4-5 hours at 37C. Take a 1 mL sample, centrifuge at top speed in a minicentrifuge, and resuspend in 100 μL of SDS-PAGE sample buffer for gel analysis.   Cool cultures to 18C.  Induce expression with IPTG to a final concentration of 0.5 mM.  Express protein overnight at 18C.  In the morning, take a 1 mL sample, centrifuge at top speed in a minicentrifuge, and resuspend in 100 μL of SDS-PAGE sample buffer for gel analysis.  Harvest bacteria by centrifugation (5000 rpm in SLA-3000 rotor, 4C, 15 minutes).  Wash pellets in PBS, transfer to a 50 mL conical tube and pellet again (5000 rpm, SLA-600 rotor, 4C, 15 minutes).  Pellets can be frozen at -80C or processed immediately.

 

Protein Purification:

 

At all steps of this purification, I take 100 μL samples, add 100 μL of SDS-PAGE sample buffer and run on a gel after the prep for gel analysis.

 

Thaw pellets on ice.  Resuspend the pellets in ice cold lysis buffer:

1 X Binding Buffer

To binding buffer, add:

0.2 mg/mL Lysozyme

1mM PMSF (from 100mM stock)

0.5X LPC

0.5% NP-40 (from 20% stock)

(I usually make ~50 mL per pellet, so I have plenty to both resuspend the pellet and to equilibrate the Ni-NTA beads).

 

Resuspend the pellets in 20 mL lysis buffer via dounce homogenization.  Incubate the bacterial mixture at room temperature for 20 minutes to allow the lysozyme to work.  Sonicate the lysates at 50% duty cycle, 2-3 output until the bacteria is no longer goopy.  Spin the sonicated material at 15,000 rpm for 60 minutes at 4C in the SS-34 rotor.  The soluble material will be in the supernatant. 

 

Combine the sonication supernatants from both preps and incubate together (I usually put the combined lysates in a 50 mL conical tube) at 4C, rotating for 1 hour.  This step allows the recombinant CPC to form. 

 

While the complex formed, I wash Ni-NTA agarose beads (from Qiagen) 2X in TBS, and 1X into the lysis buffer.  After an hour of mixing, the lysate containing CPC is then applied to the beads, and incubated at 4C, rotating for 1-2 hours.  The 6-His tag on Aurora B should bind the nickel beads.  After 1-2 hours of incubation, the beads are gently spun out of the lysate (3000 rpm in our Jouan table top centrifuge).  The lysate is decanted off the beads and saved (frozen in liquid nitrogen and stored at -80C until we are certain that the protein bound to beads).

 

Wash the beads 5X in 1X Wash Buffer.  After the 5th wash, transfer beads to a 2 mL eppendorf tube.  Spin beads gently at 3000 rpm in a minicentrifuge to remove residual buffer. 

 

Elute protein by applying 1 bed volume equivalent of 1X Elution Buffer  to bead, mixing gently by inversion to resuspend the beads in the buffer, and then gently spinning at 3000 rpm for 1 minute to collect elution.  Remove eluate to fresh tubes and store samples on ice.  Monitor elution profile by Bradford assay (we use a 96-well dish, add 150 μL of 1X Bradford reagent to 8-10 wells, and add 3-5 μL of an elution to a well, and watch for the protein peak). 

 

Protein can then either be dialyzed into a buffer of choice or run over gel filtration as an additional purification step. 

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia