Purification and immunological characterization of a recombinant trimethylflavonol 3'-O-methyltransferase

Article abstract of DOI:10.1016/S0031-9422(98)00138-1

A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysosplenium americanum was expressed in Escherichia coli Top 10 and the recombinant protein was purified to near homogeneity by affinity chromatography on chelation resin and gel filtration on Superose 12 columns. The purified protein was enzymatically active as a 42 kDa monomer and exhibited strict specificity for position 3' of 3,7,4'-tri methylquercetin. It did not accept the mono- or dimethyl analogs, the parent aglycone quercetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of polymethylated flavonol synthesis in this plant. The K(m) values of the enzyme for 3,7,4'-tri methylquercetin as substrate and S-adenosyl-L-methionine as co-substrate were 7.2 and 20 μM, respectively. The enzyme activity was strongly inhibited by both Ni²⁺ and p-chloromercuribenzoate and was restored by the addition of EDTA or β-mercaptoethanol, respectively. Antibodies raised against the F3'OMT recombinant protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS-poly-acrylamide gels of protein extracts prepared from various sources. This implies a high degree of structural similarity among these enzymes that is also corroborated by their hydropathy profiles.

Full text of DOI:10.1016/S0031-9422(98)00138-1

Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 208Ð214\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S9920Ð8311"87#99027Ð0
PURIFICATION AND IMMUNOLOGICAL CHARACTERIZATION OF
A RECOMBINANT TRIMETHYLFLAVONOL 2?!O!
METHYLTRANSFERASE
IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY
JACYNTHE SEGUIN\ INGRID MUZAC and RAGAI K[ IBRAHIMꢀ
Plant Biochemistry Laboratory\ Department of Biology\ Concordia University\ 0344 De Maisonneuve Boulevard West\
Montreal\ Que[\ Canada H2G 0M7
"Received 02 October 0886#
Key Word Index*Chrysosplenium americanum^ Saxifragaceae\ ~avonol O!methyltransferase^
heterologous expression^ puri_cation^ antibody production^ properties[
Abstract*A ~avonol O!methyltransferase cDNA clone "pF2?OMT# from Chrysosplenium americanum was
expressed in Escherichia coli Top 09 and the recombinant protein was puri_ed to near homogeneity by a.nity
chromatography on chelation resin and gel _ltration on Superose 01 columns[ The puri_ed protein was
enzymatically active as a 31 kDa monomer and exhibited strict speci_city for position 2? of 2\6\3?!tri!
methylquercetin[ It did not accept the mono! or dimethyl analogs\ the parent aglycone quercetin or the
phenylpropanoids\ ca}eic and 4!hydroxyferulic acids as substrates^ thus indicating its involvement in the later
steps of polymethylated ~avonol synthesis in this plant[ The Km values of the enzyme for 2\6\3?!tri!
methylquercetin as substrate and S!adenosyl!L!methionine as co!substrate were 6[1 and 19 mM\ respectively[
The enzyme activity was strongly inhibited by both Ni1¦ and r!chloromercuribenzoate and was restored by
the addition of EDTA or b!mercaptoethanol\ respectively[ Antibodies raised against the F2?OMT recombinant
protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS!poly!
acrylamide gels of protein extracts prepared from various sources[ This implies a high degree of structural
similarity among these enzymes that is also corroborated by their hydropathy pro_les[ Þ 0887 Elsevier Science
Ltd[ All rights reserved
INTRODUCTION
but also modi_es their solubility and\ hence\ increases
their antimicrobial activity ð5Ł[ In addition\ O!methy!
lation of ~avonoids\ such as quercetin\ is believed to
reduce their mutagenicity and\ thus\ prevents tumor
formation in mammalian tissues ð6Ł[
Chrysosplenium americanum\ a saxifragaceous semi!
aquatic weed\ accumulates a variety of tetra! to penta!
O!methylated ~avonol glucosides ð7Ł[ Their synthesis
has been shown to be catalyzed by a family of _ve\
substrate!speci_c and position!oriented O!methyl!
transferases "OMTs^ E[C[ 1[0[0[5x# and two distinct
O!glucosyltransferases "E[C[ 1[3[0[x# ð8Ł[ The local!
ization of these metabolites within the apoplastic
cellular compartment of Chrysosplenium leaves ð09\
00Ł suggested their role as protective compounds
against invasion by microorganisms ð01Ł[
Flavonoid compounds are natural constituents with
an ubiquitous distribution in the plant kingdom[
Apart from their frequently implied functions as UV
protectants and insect!attracting ~ower pigments\
~avonoid compounds have been suggested to play
other roles\ such as the regulation of auxin transport
ð0Ł\ signalling molecules in plantÐsymbiont inter!
actions ð1Ł\ determinants of pollen tube growth and
pollen functionality ð2Ł and as precursors of phy!
toalexins following microbial attack ð3\ 4Ł[ Enzymatic
methylation of ~avonoids involves the transfer of the
methyl group of S!adenosyl!L!methionine "AdoMet#
to the hydroxyl group of a ~avonoid acceptor\ with
the formation of its methyl ether derivative and S!
adenosyl!L!homocysteine as products[ O!Methy!
lation of ~avonoid compounds not only reduces the
chemical reactivity of their phenolic hydroxyl groups
In a previous paper ð02Ł we reported the isolation
of a cDNA clone "pFOMT2?# encoding an enzyme
which catalyzed the 2?:4?!O!methylation of partially
methylated ~avonols in C[ americanum and char!
acterized it at the molecular level[ This paper describes
the puri_cation to near homogeneity and some phy!
ꢀAuthor to whom correspondence should be sent[
208

219
J[ Sequin et al[
Fig[ 0[ "A# SDS!PAGE of the F2?OMT recombinant protein fractions from various puri_cation steps after Coomassie blue
staining] lane 0\ prestained protein standards^ lane 1\ non!induced bacterial lysate "09 mg#^ lane 2\ IPTG!induced bacterial
lysate "09 mg#^ lane 3\ NiÐNTA fraction "4 mg#^ lane 4\ Superose!01 "9[4 mg#^ lane 5\ Coomassie!stained protein Mr markers
"kDa#] lysozyme "03[3#\ trypsin inhibitor "10[4#\ carbonic anhydrase "20[9#\ ovalbumin "34[9#\ bovine serum albumin "55[1#
and phophorylase B "86[3#[ "B# Western blot analysis after SDS!PAGE and electroblotting onto nitrocellulose membrane[
The immune reaction was revealed with anti!F2?OMT antiserum as described in Section 2[ Lanes 0Ð4\ as described in "A#[
sicochemical and immunochemical characteristics of favoured copuri_cation of the bacterial proteins
the recombinant protein expressed in Escherichia coli[ through the formation of disul_de bonds with the
A polyclonal antibody to the puri_ed protein was protein of interest during cell lysis ð04Ł[ Furthermore\
produced and used to study the distribution of the the Mr of the recombinant protein "ca[ 27 kDa# is
enzyme within the plant\ as well as to investigate its 08 kDa lower than that previously reported for the
cross reactivity with OMTs from other sources[ The intact C[ americanum shoot OMTs\ which was deter!
results obtained suggest a high degree of structural mined by gel _ltration on Sepharose S!199 column
similarity among the enzyme proteins as corroborated ð05Ł[ It is possible that the F2?OMT gene product is
by their hydropathy pro_les[
produced in E[ coli in an immature form\ that would
normally be processed post!translationally in the
intact tissue[ A similar situation has been reported for
a methyltransferase of the benzylisoquinoline alkaloid
pathway\ scoulerine OMT ð06Ł[ Nevertheless\ the Mr
of the F2?OMT fusion protein is quite similar to those
reported for the alfalfa chalcone 1?!OMT "32 kDa#
RESULTS AND DISCUSSION
Expression and puri_cation of F2?OMT
Expression of the recombinant form of F2?OMT ð07Ł and iso~avone 6!OMT "30 kDa# ð08Ł recombinant
was carried out using the pTRC!His vector which gave proteins[ In contrast\ the native molecular mass of the
rise to a fusion product that possessed a six!histidine recombinant aspen\ lignin!speci_c ca}eic acid OMT
tag\ as part of a short leader sequence of the N!ter! ð19Ł and that of Clarkia breweri "iso#eugenol OMT
minus of the protein ð03Ł[ A 1 h!induction with iso! ð10Ł have been reported to be 79 and 77 kDa dimers\
propyl b!thiogalactopyranoside "IPTG# of the pTRC! as determined by native gradient!gel electrophoresis
His!F2?OMT in E[ coli Top 09 resulted in a signi_cant and gel _ltration chromatography\ respectively[
expression of an approximately 31 kDa protein\ as can
The use of pTRC!His vector made it possible to
be seen on Coomassie!stained SDS!polyacrylamide purify the fusion protein to near homogeneity by
gels ðFig[ 0"A#\ lanes 2 and 3Ł[ The molecular mass of a.nity chromatography on a NiÐNTA chelation
this protein is consistent with addition of the 2 kDa resin\ followed by gel _ltration on Superose 01 ðFig[
His!Tag leader sequence ð03Ł fused to the 26[5 kDa 0"A#\ lane 3Ł[ Both puri_cation steps resulted in a 037!
F2?OMT ð02Ł[ The calculated Mr of the active fraction fold enrichment of the enzyme speci_c activity\ with
of the fusion protein\ as determined by FPLC gel a 27[4) yield "Table 0#[ The signi_cant loss of enzyme
_ltration on a calibrated Superose!01 column\ was activity after a.nity chromatography can be attri!
estimated to be 3129[4 kDa "Fig[ 1#\ which suggests buted to the inhibiting e}ect of Ni1¦ "Table 2# which
that the enzyme protein is composed of a single poly! may leach from the column and remains in contact
peptide chain[ This _nding contradicts the previous with the enzyme protein[ However\ the addition of
suggestion ð02Ł that the native form of F2?OMT may both 09 mM EDTA and 03 mM b!mercaptoethanol
exist as a 65 kDa dimer\ which was formerly estimated to the protein extract in the steps following a.nity
by gel _ltration of the bacterial lysate in the absence of chromatography prevented further loss of enzyme
b!mercapto!ethanol[ The latter conditions may have activity[

Trimethyl~avonol 2?!O!methyltransferase
210
Fig[ 1[ Mr estimation of the native recombinant F2?OMT by chromatography on Superose!01 column calibrated with "kDa#]
"A# ribonuclease "02[6#^ "B# carbonic anhydrase "20[9# and "C# bovine serum albumin "56[9#[
Table 0[ Puri_cation of the recombinant F2?OMT proteinꢀ
Speci_c
activity
"nkat:mg#
Total
activity
"nkat#
Protein
"mg#
Yield
")#
Puri_cation Recovery
Fraction
"!fold#
")#
Crude extract
NiÐNTA column
Superose 01
04[3
9[24
9[93
9[34
18[45
55[64
5[82
09[24
1[56
099
038
27[4
−
54[6
037[2
099
1[29
9[15
ꢀThe puri_cation steps were carried out on the recombinant protein obtained from a 499!ml IPTG!induced
bacterial culture[ Enzyme assays were conducted as described in Section 2[
Speci_city of the anti!F2?OMT antibody
nodetection of other puri_ed recombinant OMTs\ as
well as the OMTs of the vegetative parts of C[ amer!
The speci_city of the antisera raised against the icanum and Serratula tinctoria[ The latter species is
Superose 01!puri_ed protein "Table 0# was veri_ed by known to accumulate 2!O!methylquercetin ð11Ł and
ELISA and further con_rmed by Western blot analy! we have veri_ed the existence of a quercetin 2!OMT
sis of the SDS!electrophoresed proteins[ Immu! activity in its leaves "unpublished data#[ The anti!
nodetection of the blotted protein fractions\ eluted F2?OMT antibody recognizes protein bands cor!
from the various puri_cation steps ðFig[ 0"B#\ lanes 1Ð responding to the expected Mr of the partially puri_ed
4Ł\ revealed that the anti!F2?OMT antiserum "0]0999 Serratula leaf ðFig[ 2"A#\ lanes 0 and 1Ł\ Chry!
dilution# reacted with a protein band migrating at the sosplenium leaf ðFig[ 2"A#\ lane 2Ł and root ðFig[ 2"A#\
position of the puri_ed enzyme ðFig[ 0"B#\ lanes 1Ð4Ł[ lane 3Ł OMTs\ as well as those of two puri_ed recom!
No protein bands could be detected when the blots binant proteins\ OMT0 and OMT1 "GenBank
were incubated with the preimmune serum "result not Accession No[ U05682# obtained from C[ americanum
shown#[ However\ it is interesting to note that the ðFig[ 2"B#\ lanes 1 and 2Ł[ The two latter OMTs\ whose
non!induced bacterial cells exhibit F2?OMT activity amino acid sequence di}ers in only 2 amino acid resi!
which amounts to about 49) of that of the IPTG! dues and share ca[ 74) amino acid similarity with the
induced cells "Table 1#\ which seems to be a charac! F2?OMT\ catalyze the 2?!O!methylation of ~avonoids
teristic feature of the E[ coli Top 09 cells grown in LB as well as the 2:4!O!methylation of ca}eic and ferulic
medium[ This result is further corroborated by the acids\ respectively\ albeit to a di}erent extent ð12Ł[
immunoblot shown in Fig[ 0"B#\ lane 1[
These results suggest epitope similarities among the
The anti!F2?OMT antibody was used for the immu! OMTs from di}erent sources\ which is not unexpected

211
J[ Sequin et al[
Table 1[ Recombinant F2?OMT activity and protein induction of non!induced and IPTG!
induced cellsꢀ
Speci_c activity "nkat:mg#
Protein induction by IPTG
Source of extract
−IPTG
¦IPTG
") of non!induced#
pTRC!His
pTRC!His:F2?OMT
9
9[01
9
9[16
24
54
ꢀThe recombinant protein was expressed in E[ coli Top 09 cells[ IPTG induction "0 mM#
was carried out for 1 h as described in Section 2[
Fig[ 2[ "A# Immunoblot detection of OMTs in soluble protein extracts "19 mg each#] Lanes 0 and 1] two cultivars of Serratula
tinctoria leaves^ lanes 2 and 3] Chrysosplenium americaum leaves roots\ respectively[ Pre!stained protein markers are shown
on the left[ "B# As in "A#\ but using the recombinant protein extracts "9[2 mg each# shown in lane 0\ F2?OMT^ lane 1\ OMT0
and lane 2\ OMT1[ The immunoreactive bands "indicated by an arrow heads# migrate at the expected Mr of 31 kDa[
in view of their similar catalytic functions and molec! 3"A# and "B#Ł[ The enzyme protein accepted neither
ular mass[ However\ it is interesting to note that the of the low methylated analogs "2!methyl! or 2\6!dime!
anti!F2?OMT antibody\ which was raised against the thylquercetin#\ the parent aglycone quercetin nor the
non!denatured protein\ did not inhibit the recom! phenylpropanoids ca}eic! or 4!hydroxyferulic acids
as substrates[ This indicates the strict substrate! and
position speci_cities of this OMT and its involvement
in the later steps of polymethylated ~avonol synthesis
in C[ americanum ð8Ł[
binant F2?OMT activity to any signi_cant extent when
it was added at varying concentrations between 099 mg
and 2 mg protein to assay mixtures containing 0Ð
199 mg of the puri_ed enzyme protein "data not
shown#[ This may be due to the fact that the epitopes
characteristic of the catalytic sites are not available to
the antibody\ thus preventing immunoprecipitation of
the enzyme protein during antigenÐantibody incu!
bation ð13Ł[
Other properties[ The pH optimum of the methy!
lation reaction was 6[7 in 49 mM phosphate bu}er[
The enzyme reaction had no absolute requirement for
divalent cations\ but was strongly inhibited in the
presence of 0 mM NiCl1 or NiSO3[ This inhibition was
fully restored by the addition of 0 mM EDTA "Table
2#[ While the thiol reagent b!mercaptoethanol had no
signi_cant e}ect on OMT activity\ r!chlo!
romercuribenzoate strongly inhibits it[ The fact that
such inhibition was restored by the addition of 03 mM
b!mercaptoethanol "Table 2# indicates the require!
ment for SH groups for catalytic activity[ These results
Physicochemical properties of recombinant F2?OMT
Substrate speci_city[ The puri_ed recombinant
F2?OMT
exhibits
speci_city
for
2\6\3?!tri!
methylquercetin as substrate for further methylation
at the 2?!position and gave rise to the expected tetra! are similar to those reported for the recombinant\
methyl derivative in the presence of ð03CŁAdoMet ðFig[ lignin!speci_c aspen OMT ð19Ł[ In fact\ the latter

Trimethyl~avonol 2?!O!methyltransferase
212
Fig[ 3[ "A# Computer enhanced image of the TLC separation of the 03C!labelled reaction products obtained with the
recombinant F2?OMT assayed with 2\6\3?!trimethylquercetin as substrate\ in presence of AdoMet[ The radioactive spot
comigrates with the 2?!methyl derivative as a reference compound[ "B# HPLC elution pro_le of 2\6\3?!trimethylquercetin as
the substrate "peak 0## and its radioactive 2?!methyl derivative as the F2?OMT reaction product "peak 1#\ coeluting with an
authentic sample of 2\6\2?\3?!tetramethylquercetin from an RP C07 semi!prep silica column\ using 74) MeOH\ 03) H1O\
0) HOAc as solvent in isocratic mode[
Table 2[ E}ects of Ni1¦ and r!CMB on F2?OMT activityꢀ
Addition
Concentration
"mM#
Relative activity$
") of control#
None "control#
−
099
29
099
099
02
NiCl1:NiSO3
0[9
0[9
9[0
0[9
03
Ni1¦¦EDTA
p!CMB%
p!CMB
0 mM pCMB¦03 mM b!mercaptoethanol
099
ꢀEnzyme assays were conducted as described in Section 2[
$Enzyme activity of the control amounted to 019 pkat:mg protein[
%Assayed in the absence of b!mercaptoethanol[
authors have recently demonstrated\ by site directed for the partially puri_ed enzyme from the intact tissue
mutagenesis\ that thiols were not required for the cata! ð05Ł[
lytic mechanism of this class of OMTs and that the
Hydropathy pro_les[ The structural similarity of the
enzyme sensitivity to inhibition may be due to the recombinant F2?OMT\ OMT0 and OMT1 is under!
reaction of cysteine thiol"s# near the sterically hin! lined by their remarkably similar hydropathy pro_les
dered surface of the active site ð19Ł[ The apparent Km "Fig[ 4#\ including the AdoMet binding region ð14Ł\ in
value of the enzyme for 2\6\3?!trimethylquercetin\ at spite of the fact that these three proteins exhibit dis!
a saturating concentration of AdoMet\ was estimated tinct substrate speci_cities[ This\ together with the
to be 6[1 mM and that for AdoMet at a saturating fact that the anti!F2?OMT antibody cross reacts with
concentration of the ~avonoid substrate was 19 mM other OMTs\ suggests that di}erences in the substrate
which are consistent with those previously reported speci_city of these three distinct OMTs are the result

213
J[ Sequin et al[
Fig[ 4[ Hydropathy pro_les generated for F2?OMT\ OMT0 and OMT1 amino acid sequences according to ð18Ł[ Positive and
negative values indicate hydrophobic "***# and hydrophilic " = = = # regions of the proteins\ respectively[ The location of
the putative AdoMet binding site is indicated with an arrow on each protein[
of relatively minor amino acid substitutions\ rather After ~owthrough\ the column was thoroughly
than alteration to major protein domains[
washed with bu}er A\ followed by a second wash
with the same bu}er containing 14 mM imidazole[
The recombinant protein was eluted with 149 mM
imidazole in bu}er A and concentrated on an Amicon
stirred cell "YM 09 membrane#\ changing to bu}er A
containing 09 mM EDTA\ 09) glycerol and 03 mM
EXPERIMENTAL
Plant material
Chrysosplenium americanum Schwein ex Hooker b!mercaptoethanol[ The concentrated sample was
"Saxifragaceae#\ a semi!aquatic weed\ was collected then chromatographed on a Superose 01 column using
from St[ Anicet "Quebec\ Canada#[ It was maintained an FPLC facility "Pharmacia#[ Protein puri_cation
in the greenhouse under conditions simulating its was monitored by enzyme assays and SDS!PAGE[
natural habitat\ with respect to light intensity "ca[
149 E = m−1 = s−0#\ temperature "04Ð06>C# and
humidity "constant running water#[
Protein extraction
Protein was routinely extracted from the plant
material at 3>C using 9[0 M sodium phosphate bu}er
Expression of the recombinant protein
Luria!Bertani "LB# medium
pH 6[7\ containing 09) PVPP "w:w#\ 0 ppm diethy!
lammonium diethyldithiocarbamate "w:v#\ 4 mM
EDTA and 03 mM b!mercaptoethanol[ After _l!
tration through miracloth and centrifugation at
09\999`\ the supernatant was desalted on a PD!09
column "Pharmacia# before being used[
containing
099 mg = ml−0 ampicillin was inoculated with E[ coli
Top 09 carrying pFOMT2? after cloning as was pre!
viously described ð02Ł[ The bacterial cells were grown
to a density of Aꢁ9[5Ð9[6 and were induced by the
addition of IPTG at a _nal concentration of 0 mM
and then harvested 1 h later[ The cells were collected
by centrifugation at 7\999` for 09 min and the pellets
were suspended in bu}er A "49 mM sodium phos!
phate\ pH 6[7\ 299 mM NaCl# containing 0 mg = ml−0
lysozyme and allowed to stand on ice for 29 min[ The
mixture was centrifuged at 09\999` for 19 min and the
clear supernatant was collected for further puri!
_cation[
Enzyme assay
The OMT assay was performed as previously
described ð05Ł using various ~avonoids as substrates
and ð03CŁAdoMet as the cosubstrate[ The reaction
products were extracted with a mixture of benzeneÐ
ethyl acetate "0]0\ v:v# and the organic phase was
analyzed for radioactivity by liquid scintillation spec!
trometry[ Product identi_cation was carried out by co!
chromatography with an authentic sample of 2\6\2?\3?!
tetramethylquercetin ð05Ł and by HPLC analysis on
an RP C07 semi!prep silica column using 74) MeOH\
03) H1O\ 0) HOAc as solvent in isocratic mode[
Puri_cation of the fusion protein
The fusion protein was _rst puri_ed by a.nity chro!
matography using NiÐnitrilo triacetic acid resin "NiÐ
NTA\ Qiagen\ U[S[A[# in a batch procedure according
to manufacturer|s speci_cations[ Brie~y\ the clear lys!
ate was incubated overnight at 3>C with the resin that
Protein determination
Protein concentration was determined by the
was previously equilibrated with bu}er A and the method of Bradford ð15Ł\ using the Bio!Rad reagent
mixture was loaded onto a 0[5 cm diameter column[ and bovine serum albumin as the standard protein[

Trimethyl~avonol 2?!O!methyltransferase
214
Molecular mass determination
4[ Dakora\ F[ D[ and Phillips\ D[ A[\ Physiolo`ical
and Molecular Plant Patholo`y\ 0885\ 38\ 0[
5[ Luckner\ M[\ Secondary Metabolism in Plants\
Animals and Microor`anisms[ Springer Verlag\
Berlin\ 0889[
The molecular mass of the native recombinant pro!
tein was estimated by gel _ltration on a calibrated
Superose 01 column[ The reference proteins used were
bovine serum albumin "56[9 kDa#\ carbonic anhydrase
"20[9 kDa# and ribonuclease A "02[6 kDa#[
6[ Zu\ B[ T[\ Ezell\ E[ H[ and Liehr\ J[ G[\ Journal
of Biolo`ical Chemistry\ 0883\ 158\ 181[
7[ Collins\ F[ W[\ De Luca\ V[\ Ibrahim\ R[ K[ and
Voirin\ B[\ Jay\ M[\ Zeitschrift fur Natur!
forschun`\ 0870\ 25c\ 629[
Ã
Antibody production
8[ Ibrahim\ R[ K[\ De Luca\ V[\ Khouri\ H[ E[\ Lat!
chinian\ L[\ Brisson\ L[ and Charest\ P[!M[\
Phytochemistry\ 0876\ 15\ 0126[
09[ Charest\ P[ M[\ Brisson\ L[ and Ibrahim\ R[ K[\
Protoplasma\ 0875\ 023\ 84[
00[ Marchand\ L[\ Charest\ P[ M[ and Ibrahim\ R[
K[\ Journal of Plant Physiolo`y\ 0876\ 020\ 228[
01[ French\ C[ J[\ Elder\ M[\ Leggett\ F[\ Ibrahim\ R[
K[ and Towers\ G[ H[ N[\ Canadian Journal of
Plant Patholo`y\ 0880\ 02\ 0[
New Zealand rabbits were immunized with 49 mg of
the Superose 01!puri_ed protein in complete Freund|s
adjuvant\ followed by two booster injections of 14 mg
each\ at 1!week intervals\ according to an established
protocol ð16Ł[ The immune reaction was monitored by
ELISA and Western blotting before collection of the
sera[
SDS!PAGE and Western blot analysis
02[ Gauthier\ A[\ Gulick\ P[ J[ and Ibrahim\ R[ K[\
Plant Molecular Biolo`y\ 0885\ 21\ 0052[
03[ Xpress SystemTM\ Version 1[9[ Invitrogen\ San
Diego\ CA[
Denaturing electrophoresis was performed accord!
ing to a standard procedure ð17Ł using 01 or 04)
acrylamide separating gels and 3) acrylamide stack!
ing gels[ Electroblotting on nitrocellulose membranes
was carried out using a Bio!Rad Transblot Semi!Dry
Blotter according to the manufacturer|s instructions[
After blocking the membranes with 2) non!fat dry
milk\ the blots were incubated with non!immune or
immune serum "0]0999 dilution#\ followed by the sec!
ondary antibody labeled with alkaline phosphatase
"0]2999 dilution#[ The immune reaction was revealed
using 4!bromo!3!chloro!2!indolyl phosphate and
nitroblue tetrazolium as substrates[
04[ QIAexpressionistTM[ Qiagen\ Santa Clara\ CA\
0881[
05[ De Luca\ V[ and Ibrahim\ R[ K[\ Archives of Bio!
chemistry and Biophysics\ 0874\ 127\ 485[
06[ Takeshita\ N[\ Fujikawa\ H[\ Mimura\ H[\
Fitchen\ J[ H[ and Yamada\ Y[\ Sato\ F[\ Plant
and Cell Physiolo`y\ 0884\ 25\ 18[
07[ Maxwell\ C[ A[\ Harrison\ M[ J[ and Dixon\ R[
A[\ Plant Journal\ 0882\ 3\ 860[
08[ He\ X[!Z[\ Reddy\ J[ T[ and Dixon\ R[ A[\ Plant
Molecular Biolo`y\ 0886 "in press#[
19[ Meng\ H[ and Campbell\ W[ H[\ Archives of Bio!
chemistry and Biophysics\ 0885\ 229\ 218[
10[ Wang\ J[ and Pichersky\ E[\ Archives of Bio!
chemistry and Biophysics\ 0887\ 238\ 042[
11[ Andary\ C[\ Gargadennec\ A[ and Cardon\ D[\
Polyphenols Communications\ 0885\ 07\ 10[
12[ Gauthier\ A[\ Gulick\ P[ J[ and Ibrahim\ R[ K[\
Archives of Biochemistry and Biophysics\ 0887\
240\ 132[
Acknowled`ements*This work was supported by
grants from the Natural Sciences and Engineering
Research Council of Canada and Fonds FCAR from
the Government of Quebec[ We wish to thank Pro!
fessor C[ Andary and Professor U[ Matern for their
generous gifts of Serratula tinctoria seeds[
13[ Larsson\ L[!I[\ Immunocytochemistry Theory and
Practice[ CRC Press\ Boca Raton\ FL\ 0877[
14[ Ibrahim\ R[ K[\ Trends in Plant Science\ 0886\ 1\
138[
15[ Bradford\ M[ M[\ Analytical Biochemistry\ 0865\
61\ 137[
16[ Lamoureux\ S[\ Vacha\ W[ E[ K[ and Ibrahim\ R[
K[\ Plant Science\ 0875\ 33\ 058[
17[ Laemmli\ U[ K[\ Nature\ 0869\ 116\ 579[
18[ Kyte\ J[ and Doolittle\ R[ F[\ Journal of Molecular
Biolo`y\ 0871\ 046\ 094[
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