Abstract
Glycosyltransferases are carbohydrate active enzymes containing catalytic modules involved in catalysing the biosynthesis of glycosidic bonds in oligo- and polysaccharides and glycoconjugates. One of the most comprehensive collections of Carbohydrate Active enZYmes is the CAZy database (http://www.cazy.org) comprising 120,000 glycosyltransferases allocated to 96 families based mainly on sequence homologies of their conserved and catalytically active domains (Cantarel et al., 2009). Interestingly, the glycosyltransferase activities of only about 1.6% of these proteins have been experimentally characterized (Lombard et al., 2014). In recent years, membrane-bound glycosyltransferases of a number of families have been shown to play a key role in the biosynthesis of plant cell-wall polysaccharides (Doblin et al., 2010; Scheller and Ulvskov, 2010; Driouich et al., 2012). They catalyze the transfer of glycosyl residues from donor nucleotide sugars to acceptors, forming the glycosidic bonds between adjacent glycosyl residues. Family 34 contains glycosyltransferases that have been shown to be involved in the biosynthesis of xyloglucans and transfer xylosyl residues to (1→4)-β-glucan chains (Keegstra and Cavalier, 2011). Our previous work suggests that Pinus radiata protein PrGT34B is a xyloglucan (1→6)-α-xylosyltransferase (Ade et al., 2014). Here, we describe a procedure for determining the xylosyltransferase activity of PrGT34B in vitro. We measured the transfer of xylose from the donor substrate UDP-xylose to different cello-oligosaccharide acceptor substrates under controlled reaction conditions. The assays include quantification of radioactively labeled reaction products and their identification by mass spectrometry. We also describe the purification, identification and quantification of the heterologously expressed recombinant protein PrGT34B in preparation for its use in the assays. This procedure may be applied to a wide range of glycosyltransferases in many different plant species.
Materials and Reagents
-
Spodoptera frugiperda cells (Sf9) (Life Technologies, InvitrogenTM, catalog number: 11496-015 )
-
Vector pFastBacHTaTM (Life Technologies, InvitrogenTM, catalog number: 10584-027 )
-
Escherichia coli (E. coli) strains DH10Bac (Life Technologies, InvitrogenTM, catalog number: 10359-016 )
-
20% ethanol
-
SDS
-
β-mercaptoethanol
-
Glycerol
-
Bromophenol blue
-
HEPES-NaOH (pH 7.0)
-
MgCl2
-
MnCl2
-
Triton X-100
-
Benzonase® nuclease (Novagen, catalog number: 70746 )
-
Protease inhibitors (complete EDTA free, protease inhibitor cocktail tablet) (Roche Diagnostics, catalog number: 0 4693159001 )
-
Imidazole
-
Cellotetraose, cellopentaose and cellohexaose (Associates of Cape Cod, Northstar BioProducts®, catalog numbers: 400402-1 , 400404-1 , 400406-1 )
-
Cellobiose (Sigma-Aldrich, catalog number: C7252 )
-
UDP-D-xylose (CarboSource Services, Complex Carbohydrate Research Center, product code: UDP-Xylose )
-
UDP-[14C-U]xylose (151.8 mCi/mmol) (PerkinElmer, catalog number: NEC543005UC )
-
TALON metal affinity resin (Takara Bio Company, Clontech, catalog number: 635501 )
-
BenchMark His-tagged protein standard (Life Technologies, InvitrogenTM, catalog number: LC5606 )
-
Precision Plus protein unstained standard (Bio-Rad Laboratories, catalog number: 161-0363 )
-
Mouse anti-His6 monoclonal antibody (GE Healthcare, product code: 27-4710-01 )
-
Horseradish peroxidase (HRP) conjugated secondary sheep anti-mouse antibody (GE Healthcare, product code: NA931-100UL )
-
SYPRO® Ruby protein gel stain (Life Technologies, Molecular Probes®, catalog number: S-21900 )
-
Ion exchange resin Dowex 1x8-200 (chloride form) (Sigma-Aldrich, catalog number: 217425 )
-
2,5-Dihydroxybenzoic acid (Sigma-Aldrich, catalog number: 85707 )
-
Starscint high flash-point LSC-cocktail (PerkinElmer)
-
PAGE loading dye (see Recipes)
-
Hypotonic lysis buffer (see Recipes)
-
Equilibration/wash buffer for IMAC (see Recipes)
-
Sample preparation buffer for IMAC (see Recipes)
-
IMAC elution buffer (see Recipes)
-
Reaction buffer for enzymatic assays (with radioactively labeled UDP-xylose) (see Recipes)
-
Reaction buffer for enzymatic assays (without radioactively labeled UDP-xylose) (see Recipes)
Equipment
-
Empty gravity-flow chromatography columns (20 ml volume) (Bio-Rad Laboratories, catalog number: 732-1010 )
-
Micro Bio-Spin chromatography columns (Bio-Rad Laboratories, catalog number: 732-6204 )
-
Amersham ECL detection system (GE Healthcare, product code: RPN2105 )
-
PVDF transfer membrane (Amersham Hybond-P) (GE Healthcare, product code: 10600023 )
-
NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific)
-
LAS-3000 Lite imager with software LAS-3000 Image Reader (Fujifilm Corporation)
-
FLA 2000 phosphorimager with software FLA 2000G (Fujifilm Corporation)
-
Rackbeta LKB Wallac (PerkinElmer, model: 1209 )
-
Voyager DETM PRO MALDI-TOF Workstation (Life Technologies, Applied Biosystems®)
Note: We believe that the reproducibility of the experiments does not depend on the precise equipment stated above but can be achieved using any other equivalent instrumentation.
Software
-
Software Multi Gauge v2.2 (Fujifilm Corporation)
-
Data Explorer (version 4.0.0.0) (Life Technologies, Applied Biosystems®)
-
Software FLA 2000G (Fujifilm Corporation)
-
Software LAS-3000 Image Reader (Fujifilm Corporation)
Procedure
-
Heterologous expression of the glycosyltransferase
Note: There are a vast number of protocols describing procedures for the heterologous expression of proteins. Successful expression of plant glycosyltransferase proteins for enzymatic analyses has been achieved in bacteria, yeast and various insect cell systems. We do not give a detailed description of the heterologous expression of protein PrGT34B, but only outline the procedure we used and give some information that may be helpful.
-
In our study (Ade et al., 2014), we expressed the xylosyltransferase
PrGT34B in Spodoptera frugiperda cells (Sf9) using an Autographa
californica nuclear polyhydrosis virus based baculovirus expression
vector system.
-
The cell line Sf9 had previously been used to
successfully express plant glycosyltransferases (Egelund et al., 2006).
Furthermore, Sf9 cells are highly susceptible to infection with Autographa californica nuclear polyhydrosis virus and the corresponding
baculovirus expression vector was shown to result in high expression
levels of the desired gene product in insect cells (Condreay and Kost,
2007).
-
The sequence of interest was cloned into vector
pFastBacHTaTM, recombinant bacmid DNA was generated in E. coli strains
DH10Bac or DH10MultiBac (Berger et al., 2004) and baculovirus was
produced and amplified in Sf9 cells following the instructions in the
manual “Bac-to-Bac Baculovirus Expression System”.
-
Sf9
suspension cultures (200 ml), with a starting concentration of 2.0 x 106 cells ml-1, were inoculated with baculovirus (1:1,000 v/v of amplified
virus stock to Sf9 cell suspension) and incubated with shaking (130 rpm,
72 h, 28 °C). Infected cells were harvested by centrifugation (500 x g,
10 min, 4 °C), and the cell pellets used for protein extraction (see
below for details).
-
For easy purification of the recombinant
protein prior to its use in enzymatic assays, we strongly suggest that
histidine tagged (6x histidine) fusion protein is expressed.
-
In
addition, many plant GTs, including xylosyltransferase PrGT34B, are
type-II membrane bound proteins with a single transmembrane domain (TMD)
close to the N-terminal end of the peptide, which acts as a signal
anchor for the protein’s localization to the Golgi apparatus via the ER.
For such proteins, we suggest expressing an N-terminus truncated
version of the protein, lacking the N-terminal TMD but containing the
complete functional domain. This may result in better solubility of the
heterologously expressed protein and hence an easier extractability from
the host cells (see below for details).
-
If the His6-tag is to
be fused to the protein’s N-terminus, the position of the N-terminal
truncation of the protein has to be chosen carefully. We truncated the
GT upstream of the most prominent stretch of charged amino acids between
the TMD and the conserved domain to allow for an optimal presentation
of the histidine-tag [please refer to Ade et al. (2014) for details on
the exact positions of protein truncation and His6 tagging].
-
Extraction of functional heterologous protein from Sf9 insect cells
-
Extract native recombinant protein PrGT34B from 8 x 108 freshly pelleted
Sf9 cells using ice-cold hypotonic lysis buffer (20 ml). Lyse the cells
on ice (40 min) and then mix the lysate on an orbital shaker (50 rpm, 2
h, 4 °C).
-
Clarify the lysate by centrifugation (14,000 x g, 5
min, 4 °C) and transfer the supernatant containing the Triton X-100
solubilized protein to a fresh tube.
-
Proceed to the purification
of the His6-tagged recombinant protein PrGT34B from the total extracted
protein as soon as possible.
Note: We kept the protein extract on
ice for no longer than 2 h before proceeding to protein purification to
avoid loss of functional protein by protein degradation, protein
precipitation or protein denaturation.
-
In addition, extract total
protein from 8 x 108 freshly pelleted Sf9 cells infected with
non-recombinant baculovirus or with baculovirus recombinant for empty
vector pFastBacHTaTM for negative control reactions required to calculate
background GT activities (see below).
-
Purification of the His6-tagged recombinant GT using immobilized metal affinity chromatography (IMAC)
Note: The TALON resin consists of Sepharose beads with a tetradentate metal chelator that holds Co2+ ions in electronegative pockets. At neutral pH, the histidine side groups of the target protein’s polyhistidine tags are unprotonated and bind to the chelated cobalt ions. Under these conditions, increasing imidazole concentrations in the elution buffer during chromatography competitively elutes bound polyhistidine-tagged protein from the resin. Purification of the GT is required to precisely determine the quantity of specific protein to be used in the enzymatic assay. Only in this way can the specific enzymatic activity of the GT be calculated.
-
Load an empty gravity-flow chromatography column with 2 ml of a 50%
(w/v) TALON metal affinity resin slurry in 20% ethanol at room
temperature.
-
After the resin has settled, apply a further frit
and wash the column with double distilled water (ddH2O) (20 ml) at room
temperature.
-
Equilibrate the TALON resin with equilibration/wash buffer (10 ml) at room temperature.
-
Mix the total protein lysate (20 ml) (see above) with ice-cold sample preparation buffer (5 ml).
-
Load the ice-cold 25 ml of soluble protein lysate, adjusted to 50 mM
HEPES-NaOH (pH 7.0), 100 mM NaCl, 5 mM imidazole, 10% glycerol, 1.6%
Triton X-100, 1.6 mM MgCl2, on to the column.
Note: The increased
ionic strength of the sample adjusted to 100 mM NaCl minimizes
non-specific binding to the resin and stabilizes the soluble protein on
the column during fractionation. The initial imidazole concentration of 5
mM increases the stringency for the affinity binding and decreases
background protein binding.
-
Wash the TALON resin with
equilibration/wash buffer (20 ml) and then elute His6-tagged protein
from the column with IMAC elution buffer (15 ml) at room temperature.
-
Capture successive fractions (1 ml) of the eluted protein and
directly store on ice. Analyze aliquots (10 µl) by standard SDS-PAGE to
determine the fraction with the highest concentration and purity of the
recombinant protein to be used in subsequent enzymatic assays.
-
Keep eluted soluble protein at 4 °C for only a short time before determining its enzymatic activity.
Note: We kept the purified protein on ice for no longer than 24 h
before using it in GT assays to avoid loss of functional protein by
protein degradation, protein precipitation or protein denaturation.
-
Identification of heterologously expressed recombinant protein PrGT34B by immunoblot analysis and peptide mass fingerprinting (PMF)
Note: Verification of the recombinant protein’s identity was achieved by a combination of immunoblot analysis of the His6-tagged protein and a mass spectrometric peptide analysis.
-
Denature an aliquot (10 µl) of the total protein extract (the total
protein extracted from 5.0 x 105 Sf9 cells) and of the IMAC purified and
eluted protein by heating in PAGE loading dye (5 min at 95 °C).
-
Separate the denatured protein on a 12% polyacrylamide gel together with
a suitable marker such as the BenchMark His-tagged protein standard.
-
Transfer the protein to a PVDF transfer membrane using any kind of suitable electrophoretic protein transfer method.
-
Detect the His6-tagged protein with a mouse anti-His6 monoclonal
antibody (3,000-fold diluted) in combination with a horseradish
peroxidase (HRP) conjugated secondary sheep anti-mouse antibody
(7,000-fold diluted) using any kind of suitable hybridization protocol.
-
Visualize chemiluminescent signals on the membrane using the
Amersham ECL detection system and any kind of suitable analysis system
such as the LAS-3000 Lite with the software LAS-3000 Image Reader.
-
Optional: In addition to identification of the His6-tagged protein in
the total protein extract and the IMAC eluted fraction by immunoblot
analysis, separate a second aliquot of the IMAC purified heterologously
expressed protein by standard SDS-PAGE. Stain the polyacrylamide gel
with Coomassie blue, excise the relevant protein band and subject to
PMF, using any kind of suitable commercial provider or academic
proteomics facility.
Note: We recommend the analysis of the purified protein once by PMF to unambiguously verify its identity.
-
On-gel quantification of the purified protein PrGT34B
Note: The purity of the His6-tagged recombinant protein after IMAC purification is not 100%. Consequently, accurate quantification of one specific protein in a protein mixture is not possible by spectrophotometric methods such as the NanoDrop ND-1000 and requires alternative quantification methods.
-
Load equal volumes (10 µl) of different dilutions (e.g. undiluted, 2, 5,
10 and 20-fold diluted) of the IMAC purified heterologously expressed
protein together with equal volumes (20 µl) of different dilutions of a
quantitative protein mass ladder such as the Precision Plus protein
unstained standard on a 12% polyacrylamide gel.
-
Separate the protein by any kind of standard SDS-PAGE protocol.
-
Stain the gel with the quantitative luminescent SYPRO Ruby protein gel stain following the manufacturer’s instructions.
-
Visualize the fluorescence signals with any kind of suitable
analysis system such as the FLA 2000 phosphorimager with FLA 2000G
software.
Note: The emission maximum of the SYPRO Ruby protein stain is 610 nm.
-
Quantify the protein bands of the previously identified specific
protein species on the gel based on a calibration curve obtained from
the known quantities of protein in the reference bands of the various
dilutions of the quantitative protein ladder using the software Multi
Gauge v2.2.
Note: The 100, 50 and 20 kDa bands of undiluted
Precision Plus protein standard (20 µl) correspond to 300, 1,500 and 300
ng of protein, respectively.

Figure 1. Quantification of IMAC purified heterologously expressed
protein by fluorometric analysis of a SYPRO Ruby stained SDS-gel.
Aliquots (10 μl) of four dilutions of the purified recombinant protein
and five different volumes of the protein mass standard Precision Plus
protein standard were separated on a 12% acrylamide gel. The gel was
stained with SYPRO Ruby protein gel stain (Molecular Probes) and the
fluorescent signals were visualised (A). Reference bands and bands to be
quantified were manually selected and the signal intensities of the
selected areas were detected as linear arbitrary units (LAU). The known
protein quantities for each selected reference band and their
background-corrected signal intensity values (LAU-BG) were used to
generate a calibration curve (B). Protein amounts in the selected sample
bands were calculated using the equation of the calibration curve. The
final concentration of recombinant protein in the sample was determined
by taking loaded volumes and dilution factors into account (C).
-
Quantitative in vitro glycosyltransferase assay for the Pinus radiata xylosyltransferase PrGT34B
General information:
-
This protocol describes how to determine the in vitro GT activity of the
xyloglucan xylosyltransferase PrGT34B by measuring, under constant
reaction conditions, the transfer of the monosaccharide moiety from a
nucleotide sugar such as UDP-xylose, used as the donor substrate, to an
acceptor substrate such as a cello-oligosaccharide.
-
An assay
temperature of 25 °C and a pH of 6.5-7.0 were found to be optimal in a
study of the activities of other GTs located in the Golgi apparatus in
P. radiata (Mast et al., 2009). Furthermore, recent literature suggests
that GT activities in the Golgi apparatus depend on a slightly acidic
environment (Reyes and Orellana, 2008). However, successful enzymatic in
vitro assays with enzymes closely related to the protein PrGT34B were
done under neutral or slightly alkaline conditions in previous studies
(Edwards et al., 1999; Faik et al., 2002; Cavalier and Keegstra, 2006).
Consequently, we used a neutral pH for all assays described in the
present protocol.
-
For all GT activity assays described in this
protocol, the protein PrGT34B was either in total Sf9 protein extract,
adjusted to a total protein concentration of 10 mg/ml with hypotonic
lysis buffer (see above), or in protein extract purified by IMAC and
adjusted to a total protein concentration of 2.5 mg/ml with IMAC elution
buffer (see above). In addition, dilutions of the extract purified by
IMAC were prepared with IMAC elution buffer (see above) containing
specific PrGT34B concentrations of 2,000, 100 and 40 µg/ml.
-
Total
protein extract contains HEPES-NaOH (pH 7.0) (50 mM), MgCl2 (2 mM),
glycerol (10%) and Triton X-100 (2%) and the IMAC purified protein
extract contains HEPES-NaOH (pH 7.0) (50 mM), NaCl (100 mM), imidazole
(50 mM) and glycerol (10%).
Note: Xyloglucan xylosyltransferase
activity of the heterologously expressed protein PrGT34B was assayed
with radioactively labeled UDP-[14C-U]xylose and cellobiose,
cellotetraose, cellopentaose or cellohexaose.
-
Mix protein solution (total protein extract or IMAC-purified extract)
(10 µl) with reaction buffer (40 µl), containing UDP-xylose,
UDP-[14C]xylose and cello-oligosaccharide as acceptor substrate.
-
Incubate (25 °C for e.g. 1, 17 or 44 h) the resulting reaction mixture
(50 µl) containing the extracted protein in HEPES-NaOH (pH 7.0) (50 mM),
MnCl2 (5 mM), MgCl2 (5 mM), UDP-xylose (1 or 4 mM), UDP-[14C]xylose (9
kBq, 54,300 dpm, 150 pmol) and cello-oligosaccharides (1 mM).
Notes:
-
These reaction conditions correspond to 50 nmol acceptor substrate
and 50 or 200 nmol unlabelled UDP-sugar donor substrate in the reaction
mixture, resulting in molar ratios of acceptor and donor of 1:1 or 1:4.
The amount of 150 pmol UDP-[14C] xylose added to the reaction mixture
corresponds to a concentration in the reaction mixture of only 3 µM,
changing the total concentration of added UDP-xylose only
insignificantly.
-
The assay can be done with any other molar ratio
between acceptor and donor substrate. Other GT activities can be
assayed using appropriate nucleotide sugars and donor substrates in the
assay mixture such as UDP-galactose and UDP-[14C]galactose as donor
substrates and manno-oligosaccharides as acceptor substrates to assay
the galactosyltransferase activity of galactomannan
galactosyltransferases.
-
Stop the reaction by heating (5 min, 95
°C), remove precipitated protein by centrifugation (10,000 x g, 2 min, 4
°C) and transfer the supernatant with the reaction products to a fresh
tube and place on ice.
-
Remove unincorporated nucleotide sugars
(UDP-xylose and UDP-[14C]xylose) from the reaction mixture by anion
exchange chromatography (AEC) using the ion exchange resin Dowex 1x8-200
(chloride form).
Note: The positively charged groups of the resin
act as strong anion exchangers that stay charged over a wide pH range
and bind negatively charged molecules such as nucleotide sugars in
exchange for the counter ion chloride.
-
Mix Dowex 1 x 8-200 resin
(1 g) with water (10 ml), allow the resin to swell over-night, wash the
resin twice with fresh water (10 ml), allow the resin to settle (10
min), discard the supernatant and add fresh water to give a 50% (w/v)
resin-water slurry.
-
Directly mix the cleared supernatant from the
assay reaction (50 µl) with the 50% (w/v) resin-water slurry (400 µl)
and incubate on an orbital shaker (50 rpm, 5 min, at room temperature).
-
Transfer the mixture to an empty Micro Bio-Spin chromatography
column fitted with a frit to retain the resin and centrifuge (700 x g, 1
min, at room-temperature).
Note: The effluent may be collected in a
fresh microcentrifuge tube. The Micro Bio-Spin column with the empty
microcentrifuge tube may be placed into a 15 ml Falcon tube with lid, so
the centrifugation can be done without the risk of contaminating the
centrifuge.
-
Mix the complete effluent (250-300 µl), containing
the uncharged reaction products with the incorporated radioactivity,
with scintillation counting fluid (Starscint high flash-point
LSC-cocktail) (1 ml).
-
Measure the incorporated radioactivity with a liquid scintillation counter for 5 min.
Note: Conversion of measured “counts per minute” (cpm) into
“disintegrations per minute” (dpm) is done automatically by the
instrument using a calibration curve that is generated by plotting
detected cpm values for external standards of known activity against
their known spectral quench parameters [SQP(E)].
-
Prepare control
incubations for enzymatic assays identical to those described above but
without protein and with protein extracts from Sf9 cells infected with
non-recombinant virus to determine background radioactivity resulting
from incomplete retention of unincorporated UDP-[14C]sugar during anion
exchange chromatography, enzyme independent degradation of UDP-sugar and
UDP-sugar hydrolyzing background activity. Treat these control
incubations identically to the samples and measure the remaining
unspecific background radioactivity in the AEC effluent.
-
Calculate the xylosyltransferase activity as follows.
Incorporation of xylose into reaction products is calculated from the
total radioactivity retained in the sample according to the formula
“amount of incorporated xylose into acceptor substrate per assay (in
pmol) = (A/B)*C”; with A representing the radioactivity retained in the
sample following anion exchange chromatography (in dpm), B representing
the total radioactivity added to the assay (in dpm) and C representing
the total amount of UDP-sugar (labeled + unlabeled) added to the assay
(in pmol).
-
GT activity can be shown as xylose incorporation per
assay (in pmol/assay) or as xylose incorporation per time (in pmol/h) if
the assays are incubated for a discrete time (e.g. 1, 17 and 44 h).
-
For assays containing a known quantity of the heterologous protein
PrGT34B, specific GT activity can be determined from the quantity (in
pmol) of xylose incorporated into acceptor substrates per time and per
quantity of specific enzyme (in pmol/h/µg).
-
Background
radioactivity detected in control assays (in dpm) may be used to
calculate the unspecific activity in a sample or may be directly
subtracted from the corresponding experimental sample to provide a
background-corrected value for the calculation of enzymatic activity.
-
Identification and characterization of PrGT34B xylosyltransferase assay reaction products by MALDI-TOF MS
Notes:
-
Most mass spectrometric methods are not quantitative unless an internal
standard is used that is in known amounts and is structurally identical
to the reaction product. Consequently, the use of matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)
described here can only be seen as a semi-quantitative method to
determine GT activity. However, this procedure not only shows GT
activity by detecting the expected reaction products but also allows the
structural characterization of all reaction products generated by the
enzyme PrGT34B.
-
MALDI-TOF MS has been proven highly efficient for
desorption and ionization of various biomolecules including
oligosaccharides (Garozzo et al., 1995; Garozzo et al., 1997).
-
Prepare the reaction mixture with extract containing the recombinant
protein (or without recombinant protein for control reactions) exactly
as described above (step 6F) but without adding radioactively labeled
UDP-xylose.
-
Incubate (25 °C for e.g. 17 h) the reaction mixture
(50 µl), containing the extracted protein in HEPES-NaOH (pH 7.0) (50
mM), MnCl2 (5 mM), MgCl2 (5 mM), UDP-xylose (1 or 4 mM), and
cello-oligosaccharides (1 mM).
Note: The incubation time was chosen sufficiently long to allow for maximum incorporation of xylose.
-
Stop the reaction by heating (5 min, 95 °C), remove precipitated
protein by centrifugation (10,000 x g, 2 min, 4 °C) and transfer the
supernatant with the reaction products to a fresh tube and place on ice.
-
Mix an aliquot of the supernatant (1 µl) with the MALDI matrix 2,5-dihydroxybenzoic acid (10 mg/ml in ddH2O) (9 µl).
-
Spot an aliquot of the sample-matrix mixture (1 µl) on a MALDI plate
and air dry to allow crystallization of analyte and matrix molecules on
the plate.
Note: It is important to minimize the glycerol
concentration in the mixture to make sure the molecules crystallize
uniformly on the plate.
-
Load the MALDI plate to a MALDI-TOF mass spectrometer such as the Voyager DETM PRO MALDI-TOF workstation.
Notes:
-
The spectrometer was calibrated using the three external standards
des-ArgI-bradykinin (m/z = 904), angiotensin I (m/z = 1296) and
[GluI]-fibrinopeptide B (m/z = 1570) diluted in 2,5-dihydroxybenzoic
acid.
-
Operate the spectrometer in the reflectron mode at an accelerating voltage of 20 kV with a delay time of 200 ns.
-
Compile mass spectra for each sample by plotting the mass (m) to
charge (z) ratio (x-axis) of all detected ion species against their
measured intensities (y-axis) using any suitable software such as the
Data Explorer (version 4.0.0.0).
Note: Compilation of each mass
spectrum should consist of the accumulated data from sufficient numbers
of laser shots (e.g. 100 laser shots).
-
Identify substrates and GT
reaction products in the mass spectrum based on the molecular weights
of their singly charged (z = 1) sodium adduct ions [M+Na]+.
Notes:
-
Because of the high NaCl concentration in the sample-matrix mixture
(> 1 mM), the dominant ion species in positive ionization mode are
the sodium adducts of the protonated molecular ions of the analyte.
-
Use the mass spectra generated for control reactions done without
enzyme to identify peaks that correlate to reaction products of the
recombinant protein PrGT34B.
-
Choose a suitable base peak intensity threshold to identify peaks that are above the noise in the mass spectrum.
Recipes
-
PAGE loading dye
SDS (1%)
β-mercaptoethanol (1 mM)
Glycerol (10%)
Bromophenol blue
-
Hypotonic lysis buffer
50 mM HEPES-NaOH (pH 7.0)
2 mM MgCl2
10% glycerol (v/v)
2% Triton X-100 (v/v)
Benzonase® nuclease (10 U/ml)
Protease inhibitors
Prepare in double distilled water (ddH2O)
-
Equilibration/wash buffer for IMAC
50 mM HEPES-NaOH (pH 7.0)
100 mM NaCl
5 mM imidazole
10 % glycerol (v/v)
Prepare in ddH2O
-
Sample preparation buffer for IMAC
50 mM HEPES-NaOH (pH 7.0)
500 mM NaCl
25 mM imidazole
10% glycerol (v/v)
Prepare in ddH2O
-
IMAC elution buffer
50 mM HEPES-NaOH (pH 7.0)
100 mM NaCl
50 mM imidazole
10% glycerol (v/v)
Prepare in ddH2O
-
Reaction buffer for enzymatic assays (with radioactively labeled UDP-xylose)
50 mM HEPES-NaOH (pH 7.0)
6.25 mM MnCl2
6.25 mM MgCl2
1.25 or 5 mM UDP-xylose
1.25 mM oligosaccharide acceptor substrate (cellobiose, cellotetraose, cellopentaose or cellohexaose)
UDP-[14C]xylose (9 kBq, 54,300 dpm, 150 pmol)
Prepare in ddH2O
-
Reaction buffer for enzymatic assays (without radioactively labeled UDP-xylose)
50 mM HEPES-NaOH (pH 7.0)
6.25 mM MnCl2
6.25 mM MgCl2
1.25 or 5 mM UDP-xylose
1.25 mM oligosaccharide acceptor substrate (cellobiose, cellotetraose, cellopentaose or cellohexaose)
Prepare in ddH2O
Acknowledgments
This work was supported by funding from the New Zealand Foundation for Research, Science and Technology, and the University of Auckland. We thank CarboSource Services (Complex Carbohydrate Research Center, Athens, GA) for the UDP-xylose and Scion Research for a doctoral stipend for Carsten P. Ade. Parts of the procedure described here were modified from Cavalier and Keegstra (2006).
References
-
Ade, C. P., Bemm, F., Dickson, J. M., Walter, C. and Harris, P. J. (2014). Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine). Plant J 78(2): 305-318.
-
Berger, I., Fitzgerald, D. J. and Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22(12): 1583-1587.
-
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Ade, C. P., Bemm, F., Dickson, J. M. J., Walter, C. and Harris, P. J. (2014).
In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein.
Bio-protocol 4(21): e1285. DOI:
10.21769/BioProtoc.1285.