given stock in milli-Q water. Preparing 500 μL of working
solution should be enough for a regular standard curve (see
Note 19).
6. IPP and DMAPP are provided by Echelon Biosciences Inc. as
Tris-ammonium salts. Prepare 4 mM stock solutions in milli-Q
water, dispense in 50 μL aliquots and store at À80
C. Prior to
use, dilute an aliquot of these substrates in water to prepare
1 mM working solutions. Leftover 1 mM solutions can be
frozen again and stored at À80
C.
7. This volume can change when testing different pH reaction
buffers. If 10Â buffers are used instead of 20Â, 20 μL should
be added.
8. Depending on the number of reactions, a mix of the first four
reagents can be prepared.
9. The amount of purified enzyme added to the reaction mixture
should be determined empirically. It is important that the
release of PPi by the enzyme tested does not surpass the activity
of the inorganic pyrophosphatase. In our case, 3 μg of the
Arabidopsis GGPPS11 enzyme also worked well in the assays.
10. Arabidopsis GGPPS11 was purified using the pET-G11 construct [31], which harbors a version of the enzyme lacking the
plastid targeting peptide and fused to an N-terminal 6x-histidine tag. The recombinant protein was produced in the E. coli
Rosetta 2 (DE) strain (Novagen, Merck KGaA, Darmstadt,
Germany). The cells were grown at 37
C in 100 mL of 2xYT
medium supplemented with the corresponding antibiotics.
When they reached an OD 600 of 0.5–0.8, 1 mM IPTG was
added. After the induction, the culture was incubated over
night at 18
C and bacterial cells were then recovered by
centrifugation. The pellet was resuspended in 10 mL of buffer
A (100 mM Tris–HCl pH 7.5, 100 mM NaCl, 10% glycerol)
supplemented with 1 mM DTT, 1 mg/mL lysozyme and one
tablet of complete protease inhibitor cocktail (Roche), and
incubated in ice for 20 min. After a brief sonication (five pulses
of 30 s, 17%), the cell lysate was centrifuged at 12,500 rpm at
4
C for 20 min. Then 2 μg/mL DNase I and 10 μg/mL
RNase A were added directly to the tube and gently mixed.
The mixture was incubated 20 min in ice and then centrifuged
again at 17,500 rpm at 4
C for 50 min. The supernatant was
filtered using a 0.2 μm filter and incubated for 2 h with 2 mL of
nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) previously
equilibrated with washing buffer (buffer A + 20 mM imidazole). After the incubation, the mixture was placed into a filter
column where the flow through is discarded and the Ni-NTA
beads are stacked with the recombinant protein attached. To
remove the unspecific proteins retained in the column five
34
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
solution should be enough for a regular standard curve (see
Note 19).
6. IPP and DMAPP are provided by Echelon Biosciences Inc. as
Tris-ammonium salts. Prepare 4 mM stock solutions in milli-Q
water, dispense in 50 μL aliquots and store at À80
C. Prior to
use, dilute an aliquot of these substrates in water to prepare
1 mM working solutions. Leftover 1 mM solutions can be
frozen again and stored at À80
C.
7. This volume can change when testing different pH reaction
buffers. If 10Â buffers are used instead of 20Â, 20 μL should
be added.
8. Depending on the number of reactions, a mix of the first four
reagents can be prepared.
9. The amount of purified enzyme added to the reaction mixture
should be determined empirically. It is important that the
release of PPi by the enzyme tested does not surpass the activity
of the inorganic pyrophosphatase. In our case, 3 μg of the
Arabidopsis GGPPS11 enzyme also worked well in the assays.
10. Arabidopsis GGPPS11 was purified using the pET-G11 construct [31], which harbors a version of the enzyme lacking the
plastid targeting peptide and fused to an N-terminal 6x-histidine tag. The recombinant protein was produced in the E. coli
Rosetta 2 (DE) strain (Novagen, Merck KGaA, Darmstadt,
Germany). The cells were grown at 37
C in 100 mL of 2xYT
medium supplemented with the corresponding antibiotics.
When they reached an OD 600 of 0.5–0.8, 1 mM IPTG was
added. After the induction, the culture was incubated over
night at 18
C and bacterial cells were then recovered by
centrifugation. The pellet was resuspended in 10 mL of buffer
A (100 mM Tris–HCl pH 7.5, 100 mM NaCl, 10% glycerol)
supplemented with 1 mM DTT, 1 mg/mL lysozyme and one
tablet of complete protease inhibitor cocktail (Roche), and
incubated in ice for 20 min. After a brief sonication (five pulses
of 30 s, 17%), the cell lysate was centrifuged at 12,500 rpm at
4
C for 20 min. Then 2 μg/mL DNase I and 10 μg/mL
RNase A were added directly to the tube and gently mixed.
The mixture was incubated 20 min in ice and then centrifuged
again at 17,500 rpm at 4
C for 50 min. The supernatant was
filtered using a 0.2 μm filter and incubated for 2 h with 2 mL of
nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) previously
equilibrated with washing buffer (buffer A + 20 mM imidazole). After the incubation, the mixture was placed into a filter
column where the flow through is discarded and the Ni-NTA
beads are stacked with the recombinant protein attached. To
remove the unspecific proteins retained in the column five
34
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n