Protein Expression and Purification of RPA70-AB (181-422)

Comments: pSV281 RPA70AB Constructed by L.Mizoue; May '02.This plasmid encodes RPA 70 subunit residues 181-422 as a fusion protein with an NT 6His tag.The tag can be cleaved off with TEV protease (recognition site is ENLFYQG)leaving "GS" at NT of protein

1.Pick a single colony [freshly transformed Rosetta BL21 (DE3)cells,(available from Novagene)or from a glycerol stock] in 2 ml LB containing 20 µg/ml Kanamycin (Kan)and 34 µg/ml of chloramphenicol (Cam)and grow cells until OD₆₀₀ (0.4~0.6)at 37 ℃.

2.Transfer 200 µl of cells into 25 ml of LB containing required amount of antibiotics and set it in the 37 ℃ shaker at 200 rpm,O/N.

3.Transfer the cells into 1 L LB containing antibiotics and incubate in the shaker at 250 rpm,at 37 ℃.

4.Grow cells until it reaches a OD₆₀₀ of 0.8.

5.Induce cells with 1 mM IPTG and grow cells for another 3~4 hours at 37 ℃.Check the expression by SDS-PAGE before proceeding for purification.

6.Harvest cells by centrifugation at 6000 rpm for 20 min and freeze at -30 or -70 ℃.

7.The next day resuspend cells in 50 mM Hepes containing 1 tablet of protease inhibitor cocktail per 3 L cells,0.3M NaCl,1% glycerol,10 mM Imidazole,pH 7.5.For cells from 1 L use 15 ml of resuspension buffer.

8.Add lysozyme to a final concentration of 0.5 mg/ml and incubate in ice for 10 min.

9.Add NP40 to a final concentration of 1% of the total volume.

10.Lyse the cells by sonication at power 5,20 s bursts and 30 s break for a total of 15 min (save some sample for SDS-PAGE)and centrifuge at 25,000 rpm for 20 min.

11.Load the supernatant in an equilibrated Ni-NTA column on the refrigerated FPLC.Use 50 mM Hepes/0.3 M NaCl/1% glycerol/1 tablet of PI per liter,pH 7.5 (Buffer A)to equilibrate the column.

12.Set-up a program that washes the column (twice the column size),and a gradient program running for 2 column size using Buffer A and Buffer B (same as Buffer A but containing 0.3 M Imidazole).

13.Pool fractions after checking them on SDS-PAGE.

TEV Protease Cleavage

Comment: TEV protease has a 6x His-tag on its NT.After the completion of the digestion reaction the protein of interest could be easily separated from TEV protease by passing the sample over Ni-NTA column.

14.Add TEV protease to the protein solution at a ratio of 1: 10 (protease:protein)and dialyze against 5 L of 25 mM phosphate,0.15 M NH₄Cl,2% glycerol pH 7.4,O/N in the cold room.Before adding protease save some undigested sample for SDS-PAGE.

15.Check the sample by SDS-PAGE for the completion of the digestion.

16.Pass the sample over Ni-NTA column equilibrated with Buffer A and collect flow through and two washes of approx two column size.

17.The final protein yields vary from 10-15 mg/L.

Note:

To prepare 15N or13℃15N labeled samples follow the same procedure except that in steps 2 and 3 M9 minimal medium has to be used.Also,use 50 ml culture instead of 25 ml in step 2.The protein yield is about half that of the rich medium.

Protocol by Arun K Alphonse-Ignatius,12/16/02

Updated on August 17,2004 by Kevin Weiss