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Protein Expression and Purification of SV40 T-ag (131-259)

Protein Expression and Purification of SV40 T-ag (131-259)

 

Comments: T antigen (131-259) is a GST-fusion protein. GST-T antigen has a tendency to form inclusion bodies. Several precautions have to be taken, both during expression and in the purification process to avoid aggregation. Always keep the temperature as described in this protocol and be gentle while sonicating the cells.

 

(i)              Pick a single colony in 2 ml LB medium containing AMP (100 mg/ml) and CAM (34 mg/ml) either from a freshly transformed BL21 (DE3) pLys (S) cells or from a glycerol stock. T antigen is in pGEX-2T as a GST fusion protein.

 

(ii)            Grow until the cells reach approximately 0.6 OD600 at 37 C (culture at 25 C for a O/N growth).

 

(iii)          Inoculate 1 ml of culture into 100 ml of LB medium containing respective antibiotics and grow at 25 C, O/N.

 

(iv)          Transfer 100 ml culture into 1 L LB medium (containing Amp and Cam) and grow the cells at 25 C until the cells reach 0.6 OD600. The lower temperature is to avoid inclusion body formation.

 

(v)            Induce cells with IPTG to a final concentration of 0.1 mM and grow for another 2 to 3 hours

 

(vi)          Harvest the bacteria by centrifugation at 6000 rpm (20 min) and resuspend cells in PBS (refer to Molecular Cloning manual for constituents) buffer containing 1 mM DTT (15 ml for 1 L culture) and store at 70 C, O/N.

 

(vii)        Thaw the cells and add lysozyme, EDTA, PMSF, leupeptin, aprotinin, and pepstatin, to a final concentration of 1mg/ml, 1 mM, 1 mM, 0.3 mg/ml, 0.3 mg/ml and 0.3 mg/ml, respectively.

 

(viii)      Incubate the cells at 4 C for 10 min and add NP-40 and MgCl2 to a final concentration of 1% and 7 mM, respectively.

 

(ix)           Sonicate cells at 20 s burst with 30 s break for 5 min at power 4 (GST can easily be denatured if sonication is slightly vigorous).

 

(x)             Centrifuge the E. Coli extract at 20, 000 rpm for 30 min, save pellet for SDS-PAGE.

 

(xi)           To remove nucleic acids add poly-ethyleneimine to the supernatant at a final concentration of 0.2% and mix by inverting the tube and incubate at 4 C for 10 min. Spin down at 25,000 rpm for 20 min to remove the precipitated nucleic acids.

 

(xii)         Add 5 ml of 50% glutathione-sepharose 4B (pre-equillibrated with Buffer A: 1x PBS containing 1% triton-X-100, 1 mM DTT and 7 mM MgCl2) slurry to the extract and incubate at 4 C with mild shaking for 30 min.

 

(xiii)       Pack the material in a column (gravity column) and collect the flow-through at a flow rate of ~1 ml/min.

 

(xiv)       Wash the column with 10 ml of Buffer-A (twice) followed by two 10 ml washes with Buffer-B (1x PBS containing, 1 mM DTT and 7 mM MgCl2), collect the washes for SDS-PAGE, leave 2 ml buffer in the column.

 

(xv)         Empty the column material with the bound GST-fusion protein into a falcon tube and add thrombin to a final concentration of 2~5 U/mg of fusion protein and incubate for 2~3 hours at 4 C with mild shaking. (Roughly estimate the protein concentration from expression level in the soluble fraction using SDS-PAGE)

 

(xvi)       Pack the material in the column and collect the flow through (contains T antigen) as well as two 5ml washes with buffer B.

 

(xvii)     Elute the bound GST protein with Buffer-B (25 mM Tris containing 10 mM reduced glutathione, pH 8.0). Save for SDS-PAGE.

 

(xviii)   Check the flow through, washes and the purified T antigen by SDS-PAGE.

 

(xix)        Final protein yield is 3~5 mg/L.

 

 

Note: To prepare 15N or labeled samples follow the same procedure except that in steps (iii) and (iv) M9 minimal medium should be used.

 

 

Arun K Alphonse-Ignatius

10/23/01