Biology Reference
In-Depth Information
PDE5
1-513
CyaB1
386-859
Adenylyl-
cyclase
PDE5
GAF A
PDE5
GAF B
MRGSHis
6
GSM
Fig. 1 Construct design of chimeric protein PDE5-GAF-CyaB1 that has been used for the high-
throughput screen. The histidine tag was used for affinity purification of the recombinant protein
malachite green have been reported using a microplate format (Cogan et al.
1999
).
In the case we describe here, the sensitivity of this method has been enhanced by the
addition of pyrophosphatase to the assay, which then generates two phosphates for
every cAMP molecule formed and so creates a highly sensitive adenylyl cyclase
assay that should be suitable for automation.
The success of a high-throughput screen depends on several parameters that
must be satisfied prior to screening. One prerequisite is a sufficient discrimination
between positive and negative results. This can be assessed by determination of the
Z
0
factor for each assay plate. The Z
0
parameter is based on the signal difference
between positive and negative controls and the standard deviation of the controls
(Zhang et al.
1999
). Second, even more important than signal intensity is a high
sensitivity for inhibition by chemicals in the library to be screened. In our case,
inhibition of activation of PDE5-GAF-CyaB1 by cGMP is to be examined. It can
be anticipated that this assay will be more sensitive for inhibition in the presence of
an intermediate cGMP concentration than in the presence of a saturating cGMP
concentration. As for PDE5-GAF-CyaB1, maximum activation of enzyme activity
is about 45-fold and so using a cGMP concentration that causes less than half-
maximal activation will still be sufficient for a high assay quality in terms of the
Z
0
factor. Third, signal intensity should be linearly dependent on enzyme concentra-
tion and reaction time. Fourth, the assay should be validated with reference
inhibitors and reveal comparable inhibition. As GAF reference inhibitors have
not been described so far, GAF inhibitory compounds identified “in house” have
been used as internal controls to monitor the sensitivity of our assays.
We describe here a high-throughput assay that satisfies the above parameters.
The enzyme used (PDE5-GAF-CyaB1) is a recombinant chimeric adenylyl
cyclase with an N-terminal hexa-histidine tag comprising the hPDE5 GAF
domain (amino acids 1-513) and the catalytic adenylyl cyclase domain from
CyaB1 from Anabaena (amino acids 386-859; gene ID 1105863, Fig.
1
). Three-
hundred milligrams of the recombinant protein was produced in several batches in
E. coli
as described and pooled (Bruder et al.
2006
). Protein purification was
optimized to achieve a high cGMP-activation factor. A phosphate calibration
curve with phosphate concentrations ranging from 0 to 4 nmol phosphate/reaction
tube was linear up to an OD
650
of 0.5 (Fig.
2a
). The malachite green assay can
detect 500 pmol phosphate in a 40
m
l sample in the presence of 75
m
MATP,which
is the planned substrate concentration. Including pyrophosphatase in the assay,
250 pmol of hydrolyzed cAMP can be detected reliably. According to our own
