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pETl la/none

BL21(DE3) codon+

Protein Expression

Several Sir2p homologs from various organisms have been expressed in a variety of vectors. Table I shows the Sir2p homologs that have been successfully expressed at high levels (at least 10 mg/liter).

Basic Protocol for Expression and Purification ofSir2-Af2

1. Inoculate 1 liter of glucose-enriched M9ZB medium (see Buffers and Solutions for Purifications, below, for all medium and buffer recipes) with 10 ml of overnight growth of Escherichia coli: BL21(DE3) (Stratagene, La Jolla, CA) in a selective medium containing carbenicillin at 0.1 mg/ml.

2. Grow the cells at 37° in carbenicillin (0.1 mg/ml). When the OD660 = 0.60.8, induce the cells with 1 mM final concentration of isopropyl-/3-D-thiogalacto-pyranoside (IPTG). Induce for 4 hr at 37°.

3. Spin the cells at 5000 rpm for 30 min at 4° in an RC-3B Sorvall (Newtown, CT) centrifuge and discard the supernatant carefully.

4. Resuspend the pellet in 50 ml of lysis buffer Af and run the cells through a microfluidizer or French press. Spin the lysate at 13,000 rpm for 30 min at 4° in a Sorvall GSA or similar rotor and carefully collect the supernatant.

5. Incubate the supernatant at 85° for 30 min, and then cool the lysate in ice water for 10 min and spin again at 13,000 rpm for 30 min at 4° in the same rotor. Collect the supernatant carefully and dialyze in buffer A.

6. Run the solution in buffer A over an NAD+ affinity column packed with Cibacron Blue beads (Pharmacia Biotech, Piscataway, NJ) and wash the column with buffer A-NaCl. Finally, elute the protein with buffer B. Dialyze the eluate in buffer C.

7. Run the solution in buffer C over a MonoQ 10/10 anion-exchange column (Pharmacia) and elute the protein in a salt gradient with buffer C-NaCl. Dialyze the protein in buffer D.

8. Run the protein in buffer D over a Superdex 75 sizing column and collect fractions of pure protein (see Fig. 4A). This protocol yields 10-20 mg/liter of culture medium.

Sir2-Af

Sir2-Tm

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