washes of 1 mL of washing buffer were performed, and finally
the protein was recovered in one tube by five elutions with
150 μL of elution buffer (buffer A + 150 mM imidazole). The
eluted sample was desalted by Thermo Scientific™ Zeba™
Desalting Columns using buffer A as exchange buffer and the
purified protein was quantified, aliquoted with glycerol 40%
and finally stored at À20
C.
11. Reagents and enzymes may be contaminated with Pi which will
interfere with the measurements. The reagents should be
tested in a standard reaction (Table 1) with no inorganic pyrophosphatase (no-pyrophosphatase control). If present, contaminating Pi should be subtracted out from the experimental
reaction measurements. In addition to the reagents, the purified enzyme may also show Pi contamination that could prevent the obtainment of conclusive data. To reduce the
contaminating Pi, the enzyme sample can be preincubated for
10 min before the addition of the substrates (e.g., IPP and
DMAPP). In this preincubation step, the reaction catalyzed by
the PNP can act as a “Pi mop” lowering the contaminating Pi
to submicromolar levels. It is recommended to always carry out
this step to avoid any possible Pi interference.
12. The concentration of IPP and DMAPP was empirically calculated to observe an increase of enzymatic activity throughout
the time using 3 μg of enzyme. We add threefold more IPP
than DMAPP since every GGPP molecule is formed from three
molecules of IPP and only one of DMAPP. The concentration
and proportion of the substrates may be determined empirically for other SC-PTs.
For the generation of pH curves we used the amount of
substrates specified in Table 1.
For kinetic parameters determination assays, the concentration of the substrates will change and so will their required
volumes. We calculated the basic kinetic parameters (V max
and K m ) for each substrate separately, designing the plate to
test different concentrations of one of the substrates fixing the
other one in 100 μM (Fig. 2). The range of concentrations
used to calculate the kinetics of the enzyme for each substrate
was: 5, 10, 20, 50, 75, 100, and 200 μM.
13. The no-substrate control must be done whenever a reagent of
the Standard Reaction changes. For example, when testing the
activity of the experimental enzyme in the presence of different
pH media, a no-substrate control must be included for each pH
(column 4 of the Fig. 2, light grey). However, for kinetic
parameters determination assays, only one no-substrate control
is needed (well A4 of the Fig. 2) since only the volume of the
substrates changes.
Determination of GGPP Synthase Activity
35
the protein was recovered in one tube by five elutions with
150 μL of elution buffer (buffer A + 150 mM imidazole). The
eluted sample was desalted by Thermo Scientific™ Zeba™
Desalting Columns using buffer A as exchange buffer and the
purified protein was quantified, aliquoted with glycerol 40%
and finally stored at À20
C.
11. Reagents and enzymes may be contaminated with Pi which will
interfere with the measurements. The reagents should be
tested in a standard reaction (Table 1) with no inorganic pyrophosphatase (no-pyrophosphatase control). If present, contaminating Pi should be subtracted out from the experimental
reaction measurements. In addition to the reagents, the purified enzyme may also show Pi contamination that could prevent the obtainment of conclusive data. To reduce the
contaminating Pi, the enzyme sample can be preincubated for
10 min before the addition of the substrates (e.g., IPP and
DMAPP). In this preincubation step, the reaction catalyzed by
the PNP can act as a “Pi mop” lowering the contaminating Pi
to submicromolar levels. It is recommended to always carry out
this step to avoid any possible Pi interference.
12. The concentration of IPP and DMAPP was empirically calculated to observe an increase of enzymatic activity throughout
the time using 3 μg of enzyme. We add threefold more IPP
than DMAPP since every GGPP molecule is formed from three
molecules of IPP and only one of DMAPP. The concentration
and proportion of the substrates may be determined empirically for other SC-PTs.
For the generation of pH curves we used the amount of
substrates specified in Table 1.
For kinetic parameters determination assays, the concentration of the substrates will change and so will their required
volumes. We calculated the basic kinetic parameters (V max
and K m ) for each substrate separately, designing the plate to
test different concentrations of one of the substrates fixing the
other one in 100 μM (Fig. 2). The range of concentrations
used to calculate the kinetics of the enzyme for each substrate
was: 5, 10, 20, 50, 75, 100, and 200 μM.
13. The no-substrate control must be done whenever a reagent of
the Standard Reaction changes. For example, when testing the
activity of the experimental enzyme in the presence of different
pH media, a no-substrate control must be included for each pH
(column 4 of the Fig. 2, light grey). However, for kinetic
parameters determination assays, only one no-substrate control
is needed (well A4 of the Fig. 2) since only the volume of the
substrates changes.
Determination of GGPP Synthase Activity
35