In order to determine enzyme parameters, for example, in a
reaction kinetic, it is required to individually capture both unmetabolized substrate (IPP) amounts, intermediates (GGPP) as well as
the final product phytoene. To achieve this, we have adopted a
butanol/heptane-based extraction protocol which is capable to
differentially extract these compounds. Moreover, incorporation
of
14
C-labeled IPP in isotope dilution allows to exactly quantify
the amounts of all components through scintillation counting.
The protocol described here applies to the enzyme combination GGPP synthase 11 (GGPS11) and PSY from Arabidopsis. We
have recently found that most of the plastid-localized GGPP
synthases are capable of providing GGPP for phytoene synthesis
by Arabidopsis PSY if coexpressed in yeast [27]. We have almost
exclusively performed in vitro assays with GGPS11 and therefore
recommend the use of this isoenzyme for the assay. In contrast, we
have successfully applied the protocol to purify and determine
enzyme activities for PSY variants from different plant sources
(e.g., maize and rice PSY1, cassava PSY1 and PSY2, tomato
PSY3), and we are therefore confident that this protocol can be
used for all plant-derived PSY versions [18, 25].
2 Materials
2.1 Recombinant
GGPP Synthase
The requirements to work with radioactive materials in your lab
must be met. Safety rules for working with radioactive substances
must be followed. Use analytical grade reagents and ultrapure
deionized water. All reagents and cellular extracts should be kept
on ice during the enzyme extraction and enzyme assay preparation
procedure (unless indicated otherwise). Waste disposal regulations
including radioactive wastes and rules for the work with bacteria
and recombinant DNA must be followed.
1. Vector containing GGPS
GGPS expression plasmids should contain a GGPP
synthase, truncated by its transit peptide as an N-terminal
fusion with a 6ÂHis-tag. C-terminal fusions might affect
GGPS activity [27]. Transit peptide predictions can be performed with chloroP [28]. Although several GGPP synthases
are able to deliver GGPP for phytoene synthesis when coexpressed in yeast [27], we have successfully worked with Arabidopsis GGPP11 (accession number At4g36810) and its
homolog from Sinapis [29] and maize for in vitro assays combined with PSY from various sources.
2. Competent BL21 E. coli cells.
3. Ice, heating block at 42
C, incubator shaker at 37
C.
42
Maurizio Camagna and Ralf Welsch
reaction kinetic, it is required to individually capture both unmetabolized substrate (IPP) amounts, intermediates (GGPP) as well as
the final product phytoene. To achieve this, we have adopted a
butanol/heptane-based extraction protocol which is capable to
differentially extract these compounds. Moreover, incorporation
of
14
C-labeled IPP in isotope dilution allows to exactly quantify
the amounts of all components through scintillation counting.
The protocol described here applies to the enzyme combination GGPP synthase 11 (GGPS11) and PSY from Arabidopsis. We
have recently found that most of the plastid-localized GGPP
synthases are capable of providing GGPP for phytoene synthesis
by Arabidopsis PSY if coexpressed in yeast [27]. We have almost
exclusively performed in vitro assays with GGPS11 and therefore
recommend the use of this isoenzyme for the assay. In contrast, we
have successfully applied the protocol to purify and determine
enzyme activities for PSY variants from different plant sources
(e.g., maize and rice PSY1, cassava PSY1 and PSY2, tomato
PSY3), and we are therefore confident that this protocol can be
used for all plant-derived PSY versions [18, 25].
2 Materials
2.1 Recombinant
GGPP Synthase
The requirements to work with radioactive materials in your lab
must be met. Safety rules for working with radioactive substances
must be followed. Use analytical grade reagents and ultrapure
deionized water. All reagents and cellular extracts should be kept
on ice during the enzyme extraction and enzyme assay preparation
procedure (unless indicated otherwise). Waste disposal regulations
including radioactive wastes and rules for the work with bacteria
and recombinant DNA must be followed.
1. Vector containing GGPS
GGPS expression plasmids should contain a GGPP
synthase, truncated by its transit peptide as an N-terminal
fusion with a 6ÂHis-tag. C-terminal fusions might affect
GGPS activity [27]. Transit peptide predictions can be performed with chloroP [28]. Although several GGPP synthases
are able to deliver GGPP for phytoene synthesis when coexpressed in yeast [27], we have successfully worked with Arabidopsis GGPP11 (accession number At4g36810) and its
homolog from Sinapis [29] and maize for in vitro assays combined with PSY from various sources.
2. Competent BL21 E. coli cells.
3. Ice, heating block at 42
C, incubator shaker at 37
C.
42
Maurizio Camagna and Ralf Welsch