19. FLuc wash buffer: 20 mM Tris–HCl, 10 mM imidazole,
200 mM NaCl, 10 mM DTT, 2 mM PMSF, 0.5Â Roche
Protease Inhibitor cocktail, 20 mM nicotinamide, pH 8.0,
0.1 mg/mL Lysozyme, DNase I.
20. FLuc-store buffer: 20 mM Tris pH 8, 50 mM NaCl,
10 mM DTT.
21. Microfluidizer (110S, Hyland SCIENTIFIC).
22. Poly-Prep
® Chromatography Columns (9 cm, 2 mL bed volume, Bio-Rad).
23. Amicon Ultra-15 (10 kDa, 30 kDa, Merck).
24. Superdex 200 column (HiLoad 26/600, GE Healthcare).
25. NanoDrop (ND-1000, Peqlab).
26. Microtiter plate reader (FLUOstar Omega, BMG Labtech).
3 Methods
The following three sections outline the necessary steps for the
creation, selection, and characterization of a KDAC mutant library.
We are using E. coli KDAC CobB as an example. CobB is a multifunctional enzyme able to reverse various lysine modifications such
as acetylation, crotonylation, propionylation or butyrylation. In
this example, we decided to remove its decrotonylation activity by
directed evolution.
3.1 Creation of KDAC
Mutant Libraries by
Inverse PCR
The active site residues of sirtuins were randomized by three rounds
of enzymatic inverse PCR [27]. Positions in this example were
chosen based on the crystal structure of CobB (pdb-file: 1S5P
[28]) close to the bound acetyl-lysine head group (~7 A ˚ radius,
see Fig. 1).
3.1.1 Inverse PCR
Primers contain randomized bases (NNK) at the sites of interest
and recognition sites for Bsa I-HF to allow scar less ligation for
plasmid circularization (Fig. 2).
1. Set up several PCRs in parallel as indicated in Table 2 (see Notes
2 and 3):
2. Run PCR according to the program detailed in Table 3.
3. Add 1 μL of Dpn I (20 U μL
À1 , NEB) to each 50 μL PCR.
Incubate for 1 h, 37
C.
4. Purify PCR product by QIA Quick PCR Purification Kit or by
Gel purification kit (see Fig. 3).
5. Bsa I-HF digestion of the PCR products for 2 h at 37
C
(Table 4).
6. Purify PCR product by QIA Quick PCR Purification Kit and
elute in 50 μL.
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Martin Spinck et al.
200 mM NaCl, 10 mM DTT, 2 mM PMSF, 0.5Â Roche
Protease Inhibitor cocktail, 20 mM nicotinamide, pH 8.0,
0.1 mg/mL Lysozyme, DNase I.
20. FLuc-store buffer: 20 mM Tris pH 8, 50 mM NaCl,
10 mM DTT.
21. Microfluidizer (110S, Hyland SCIENTIFIC).
22. Poly-Prep
® Chromatography Columns (9 cm, 2 mL bed volume, Bio-Rad).
23. Amicon Ultra-15 (10 kDa, 30 kDa, Merck).
24. Superdex 200 column (HiLoad 26/600, GE Healthcare).
25. NanoDrop (ND-1000, Peqlab).
26. Microtiter plate reader (FLUOstar Omega, BMG Labtech).
3 Methods
The following three sections outline the necessary steps for the
creation, selection, and characterization of a KDAC mutant library.
We are using E. coli KDAC CobB as an example. CobB is a multifunctional enzyme able to reverse various lysine modifications such
as acetylation, crotonylation, propionylation or butyrylation. In
this example, we decided to remove its decrotonylation activity by
directed evolution.
3.1 Creation of KDAC
Mutant Libraries by
Inverse PCR
The active site residues of sirtuins were randomized by three rounds
of enzymatic inverse PCR [27]. Positions in this example were
chosen based on the crystal structure of CobB (pdb-file: 1S5P
[28]) close to the bound acetyl-lysine head group (~7 A ˚ radius,
see Fig. 1).
3.1.1 Inverse PCR
Primers contain randomized bases (NNK) at the sites of interest
and recognition sites for Bsa I-HF to allow scar less ligation for
plasmid circularization (Fig. 2).
1. Set up several PCRs in parallel as indicated in Table 2 (see Notes
2 and 3):
2. Run PCR according to the program detailed in Table 3.
3. Add 1 μL of Dpn I (20 U μL
À1 , NEB) to each 50 μL PCR.
Incubate for 1 h, 37
C.
4. Purify PCR product by QIA Quick PCR Purification Kit or by
Gel purification kit (see Fig. 3).
5. Bsa I-HF digestion of the PCR products for 2 h at 37
C
(Table 4).
6. Purify PCR product by QIA Quick PCR Purification Kit and
elute in 50 μL.
324
Martin Spinck et al.