3.3.3 Characterization
of Isolated Mutants
For testing a large number of mutants for a desired property,
KDACs partially purified by Ni-NTA (Subheading 3.3.2, steps 1–
14) or even cell lysates can be used if mutants can be expressed in an
E. coli ΔcobB strain (e.g., DH10B ΔcobB ΔpyrF). The assay is
exceptionally sensitive, so that even small contaminations with
KDACs such as E. coli CobB will produce high background signals.
All isolated mutants are expressed, and activity for different modifications is assessed. Eventually, the desired KDACs should be
purified to homogeneity by Ni-NTA affinity and gel filtration chromatography for full characterization.
1. Prepare cell lysate (Subheading 3.3.2, steps 1–10), Ni-Eluate
(steps 1–14), or fully purified KDACs (steps 1–18) of all
mutants and a wild-type control.
2. Estimate protein concentration by SDS-PAGE or nano drop.
3. Set up reactions in 96-well PCR plates in triplicate for every
lysine modification as following: 0.2 mg/mL KDAC, 2 mM
NAD
+ , 10 μL Luciferase eluate (1:125 to 1:8000 dilution,
depending on the signal strength, at least 100-fold over background) in KDAC reaction buffer to a final volume of 50 μL.
4. Incubate at 30
C for 30 min with 50
C lid temperature in a
thermocycler.
5. Transfer 40 μL of the reaction mixture to a 96-well microtiter
plate.
6. Add 40 μL of 2Â Luciferase Buffer.
7. Measure luminescence using a Microplate reader.
8. Normalize activity to the signal of the wild-type.
9. Mutants with enhanced selectivity and/or activity are selected
for further analysis.
10. Perform steps 1–8 with various concentrations of mutant and
wild-type enzyme (between 1 nM and 16 μM).
11. Activity can be compared in the linear region of the obtained
curve for each individual substrate.
4 Notes
1. The primer needs to be as defined as possible. Prime fragments
can lead to PCR artifacts and increase side products. Ultimately
this decreases library quality and increases the effort in
creating it.
2. Total volume is adjusted to required diversity. For example,
4 Â 10
7 mutants approximately 10–16 PCRs are required,
depending on the yield and ligation efficiency.
334
Martin Spinck et al.
of Isolated Mutants
For testing a large number of mutants for a desired property,
KDACs partially purified by Ni-NTA (Subheading 3.3.2, steps 1–
14) or even cell lysates can be used if mutants can be expressed in an
E. coli ΔcobB strain (e.g., DH10B ΔcobB ΔpyrF). The assay is
exceptionally sensitive, so that even small contaminations with
KDACs such as E. coli CobB will produce high background signals.
All isolated mutants are expressed, and activity for different modifications is assessed. Eventually, the desired KDACs should be
purified to homogeneity by Ni-NTA affinity and gel filtration chromatography for full characterization.
1. Prepare cell lysate (Subheading 3.3.2, steps 1–10), Ni-Eluate
(steps 1–14), or fully purified KDACs (steps 1–18) of all
mutants and a wild-type control.
2. Estimate protein concentration by SDS-PAGE or nano drop.
3. Set up reactions in 96-well PCR plates in triplicate for every
lysine modification as following: 0.2 mg/mL KDAC, 2 mM
NAD
+ , 10 μL Luciferase eluate (1:125 to 1:8000 dilution,
depending on the signal strength, at least 100-fold over background) in KDAC reaction buffer to a final volume of 50 μL.
4. Incubate at 30
C for 30 min with 50
C lid temperature in a
thermocycler.
5. Transfer 40 μL of the reaction mixture to a 96-well microtiter
plate.
6. Add 40 μL of 2Â Luciferase Buffer.
7. Measure luminescence using a Microplate reader.
8. Normalize activity to the signal of the wild-type.
9. Mutants with enhanced selectivity and/or activity are selected
for further analysis.
10. Perform steps 1–8 with various concentrations of mutant and
wild-type enzyme (between 1 nM and 16 μM).
11. Activity can be compared in the linear region of the obtained
curve for each individual substrate.
4 Notes
1. The primer needs to be as defined as possible. Prime fragments
can lead to PCR artifacts and increase side products. Ultimately
this decreases library quality and increases the effort in
creating it.
2. Total volume is adjusted to required diversity. For example,
4 Â 10
7 mutants approximately 10–16 PCRs are required,
depending on the yield and ligation efficiency.
334
Martin Spinck et al.