7. Supplement the supernatant with 500 μL Ni-NTA-beads.
8. Incubate the beads for 2 h with agitation at 4
C.
9. Wash the beads with 50 mL FLuc-wash buffer and elute bound
proteins in 2 mL FLuc-wash buffer supplemented with
200 mM imidazole.
10. Exchange buffer to FLuc-store buffer using a centrifugal filter.
11. Use the Fluc as deacetylase substrate (see Note 12).
3.3.2 Purification
of CobB
1. Transform E. coli ΔpyrF ΔcobB with pBK-His 6 -CobB and
pPylT-Ura3 by a method of choice (see Note 13).
2. Setup 20 mL overnight culture in LB medium (containing
15 μg/mL tet, 50 μg/mL kan) from a single colony.
3. Inoculate 1L LB medium (containing 15 μg/mL tet, 50 μg/
mL kan) with 10 mL overnight culture.
4. Grow to OD 0.3–0.5 at 37
C and 200 rpm.
5. Decrease temperature to 30
C and induce protein expression
by addition of arabinose (0.2% final).
6. Harvest cells by centrifugation (6000 Â g, 20 min) after 16 h of
expression.
7. Wash the cell pellet with PBS and store at À20
C.
8. Suspend the cell pellet in 25 mL CobB wash buffer supplemented with lysozyme (0.1 mg/mL) and DNase (~0.1 mg).
9. Lyse cells by passing through a microfluidizer 2–3 times.
10. Pellet cell debris by centrifugation and decant supernatant into
a Falcon tube.
11. Add 500 μL Ni-NTA resin to the supernatant, incubate for 1 h
with agitation.
12. Pour the suspension into a plastic column with frit.
13. Wash with 50 mL CobB wash buffer.
14. Elute protein in 2 mL Elution buffer.
15. Concentrate fractions using a centrifugal filter and exchange
buffer by diluting twice with gel filtration buffer.
16. Apply the concentrated fractions to a Superdex 200 column
equilibrated with gel filtration buffer (2.5 mL/min, 4
C).
17. Collect 4 mL fractions while monitoring protein concentration
at 280 nm.
18. Analyze protein containing fractions by SDS-PAGE, pool, and
concentrate by ultrafiltration.
19. Flash freeze protein in liquid nitrogen and store at À80
C.
A Directed Evolution System for Lysine Deacetylases
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8. Incubate the beads for 2 h with agitation at 4
C.
9. Wash the beads with 50 mL FLuc-wash buffer and elute bound
proteins in 2 mL FLuc-wash buffer supplemented with
200 mM imidazole.
10. Exchange buffer to FLuc-store buffer using a centrifugal filter.
11. Use the Fluc as deacetylase substrate (see Note 12).
3.3.2 Purification
of CobB
1. Transform E. coli ΔpyrF ΔcobB with pBK-His 6 -CobB and
pPylT-Ura3 by a method of choice (see Note 13).
2. Setup 20 mL overnight culture in LB medium (containing
15 μg/mL tet, 50 μg/mL kan) from a single colony.
3. Inoculate 1L LB medium (containing 15 μg/mL tet, 50 μg/
mL kan) with 10 mL overnight culture.
4. Grow to OD 0.3–0.5 at 37
C and 200 rpm.
5. Decrease temperature to 30
C and induce protein expression
by addition of arabinose (0.2% final).
6. Harvest cells by centrifugation (6000 Â g, 20 min) after 16 h of
expression.
7. Wash the cell pellet with PBS and store at À20
C.
8. Suspend the cell pellet in 25 mL CobB wash buffer supplemented with lysozyme (0.1 mg/mL) and DNase (~0.1 mg).
9. Lyse cells by passing through a microfluidizer 2–3 times.
10. Pellet cell debris by centrifugation and decant supernatant into
a Falcon tube.
11. Add 500 μL Ni-NTA resin to the supernatant, incubate for 1 h
with agitation.
12. Pour the suspension into a plastic column with frit.
13. Wash with 50 mL CobB wash buffer.
14. Elute protein in 2 mL Elution buffer.
15. Concentrate fractions using a centrifugal filter and exchange
buffer by diluting twice with gel filtration buffer.
16. Apply the concentrated fractions to a Superdex 200 column
equilibrated with gel filtration buffer (2.5 mL/min, 4
C).
17. Collect 4 mL fractions while monitoring protein concentration
at 280 nm.
18. Analyze protein containing fractions by SDS-PAGE, pool, and
concentrate by ultrafiltration.
19. Flash freeze protein in liquid nitrogen and store at À80
C.
A Directed Evolution System for Lysine Deacetylases
333