Protein Expression and Purification 73 (2010) 90–98
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
High-level expression of barley b-D-glucan exohydrolase HvExoI
from a codon-optimized cDNA in Pichia pastoris
Sukanya Luang a, Maria Hrmova b,*, James R. Ketudat Cairns a,**
a
b
School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
Australian Centre for Plant Functional Genomics, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
a r t i c l e
i n f o
Article history:
Received 13 March 2010
and in revised form 12 April 2010
Available online 18 April 2010
Keywords:
Catalytic properties
Hordeum vulgare
N-deglycosylation
Substrate specificity
Transglycosylation
a b s t r a c t
The native b-D-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the
first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding
of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression
of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20 °C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70–
110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in
native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-b-D-glucan, laminarin and lichenans. The catalytic efficiency value kcat/KM of rHvExoI
with barley (1,3;1,4)-b-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose,
cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl b-D-glucoside and barley (1,3;1,4)-b-D-glucan. Overall, the biochemical properties of rHvExoI were similar to
those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates
of certain b-linked glucosides.
Ó 2010 Elsevier Inc. All rights reserved.
Introduction
Glycoside hydrolases (EC 3.2.1.) are widely distributed in living
organisms. These enzymes hydrolyse glycosidic linkages between
two or more carbohydrates or between a carbohydrate and a
non-carbohydrate moiety. Based on amino acid sequence similarities, catalytic mechanisms and structural features, the glycoside
hydrolase family GH3 is one of 115 glycoside hydrolase families
currently listed in the CAZy database (http://www.cazy.org/) [1].
The GH3 enzymes are more frequently represented in bacteria,
plants and fungi, than in archaea and mammals. The GH3 family
includes catalytic proteins with b-D-glucosidase (EC 3.2.1.21), xylan 1,4-b-D-xylosidase (EC 3.2.1.37), b-N-acetylhexosaminidase
(EC 3.2.1.52), exo-b-D-glucanase (EC 3.2.1.-) and a-L-arabinofuranosidase (EC 3.2.1.55) activities. The predicted functions of the
GH3 enzymes involve: (i) the biodegradation and assimilation of
oligo- and polysaccharides [2–5], (ii) modification of bacterial macrolide antibiotics and other toxic plant compounds [6,7], and (iii)
turnover of cell wall components [8–15]. The biochemical and bio-
* Corresponding author. Fax: +61 883037102.
** Corresponding author. Fax: +66 44224185.
E-mail addresses: maria.hrmova@adelaide.edu.au (M. Hrmova), cairns@sut.ac.th
(J.R. Ketudat Cairns).
1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2010.04.011
physical properties of various GH3 enzymes have previously been
described [16–29]. Some GH3 enzymes exhibit broad substrate
specificity, such as the b-D-glucosidases BGL1 from Pichia etchellis
and Saccharomycopsis fibuligera [30,31], Gbg1 from Agrobacterium
tumefaciens [32], and bglB from Thermotoga neapolitana [33] and
enzymes with a-L-arabinofuranosidase and b-D-xylosidase activities, such as ARA-I/XYL from barley (Hordeum vulgare), XarB from
Thermoanaerobacter ethanolicus and MsXyl1 from alfalfa roots
[34–36]. Therefore, the identification of natural substrates of GH3
enzymes based on their hydrolytic reactions, and assignments of
the biological functions of these enzymes are often highly
conjectural.
One of the most intensely studied enzymes in the GH3 family is
a barley b-D-glucan exohydrolase, isoform ExoI [37], here designated as HvExoI. Based on substrate specificity and gene expression studies, it has been suggested that this enzyme might be
involved in the turnover or modification of cell walls during the
elongation of coleoptiles [9,15]. The enzyme is able to hydrolyse
a variety b-D-glucosidic linkages [37,38]. HvExoI was the first
GH3 enzyme for which a crystal structure was determined [39].
The 3D structure consists of an NH2-terminal (b/a)8 barrel domain
and a COOH-terminal (a/b)6 sandwich domain [39]. Two catalytic
amino acid residues were identified in the active site and these
are the catalytic nucleophile Asp285, located in the conserved
S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
SDW motif of the first domain, and the acid/base Glu491, positioned in the second domain [40,41]. The active site of HvExoI is located at the interface between the two domains and a glucose
molecule was found at the 1 subsite in the native crystal structure, bound predominantly through hydrogen bonds to charged
amino acid residues [39].
The catalytic mechanism and structural basis of substrate specificity of HvExoI have been investigated by kinetic and crystallographic studies with substrate analogues and inhibitors [41–44].
Based on the premise that enzymes in the same family possess
similar structures and catalytic mechanisms, HvExoI was also used
as a structural model to predict the architectures of other GH3
members [45]. The catalytic mechanism of other GH3 enzymes
has also been studied by site-directed mutagenesis with enzymes
for which a crystal structure has yet to be determined, such as a
b-D-glucosidase from Flavobacterium meningosepticum [46,47].
However, similar mutagenesis studies with HvExoI have yet to be
conducted due to lack of an appropriate recombinant expression
system. It is expected that the catalytic mechanism of HvExoI
may be more precisely understood, if the roles of active site residues suggested from the structural studies could be confirmed by
a site-directed mutagenesis approach.
Although many plant glycoside hydrolases have been expressed
in bacteria, not every glycoside hydrolase could be expressed in a
prokaryotic system in an active form. The yeast Pichia pastoris has
proven to be an effective eukaryotic host in many other instances
(e.g. [48,49]). P. pastoris is a methylotrophic yeast, which can produce large amounts of recombinant proteins by methanol induction
of the alcohol oxidase 1 (AOX) promoter, and has been shown to
effectively synthesize eukaryotic post-translationally modified proteins [50,51]. P. pastoris has similar molecular genetics to Saccharomyces cerevisiae, but researchers have achieved much higher yields
of plant glycoside hydrolases, such as barley a-amylase [48] and Thai
rosewood b-D-glucosidase [52,53], in P. pastoris than S. cerevisiae.
Here, we report the recombinant expression of the HvExoI isoenzyme (rHvExoI) from barley in various expression systems. This
is the first successful recombinant expression of an active form of a
plant enzyme from the GH3 family that as of April 2010 contains
nearly 3000 entries. We further describe the substrate specificity
and biochemical properties of rHvExoI expressed in P. pastoris,
and compare these characteristics with those of the native HvExoI
enzyme.
Materials and methods
Chemicals, reagents and expression plasmids
The substrates 4-nitrophenyl-b-D-glucopyranoside (4NPGlc)1,
4-nitrophenyl-b-D-galactopyranoside, 4-nitrophenyl-b-D-xylopyranoside, 4-nitrophenyl-b-D-fucopyranoside, 4-nitrophenyl-a-L-arabinopyranoside, laminarin (Laminaria digitata), orcinol, bovine serum
albumin (BSA) and the glucose diagnostic kit were purchased from
Sigma (St. Louis, MO, USA). The sources of other substrates have
been described previously [38]. For recombinant plasmid construction, the previously described bacterial pET32a/DEST [54] and yeast
pPICZaBNH8 [53] expression vectors were used, while the pPICZaBNH8/DEST vector was created by inserting the Gateway conversion cassette C from Invitrogen (Carlsbad, CA, USA) into the SnaBI
site in pPICZaBNH8.
1
Abbreviations used: 4NP, 4-nitrophenyl; 4NPGlc, 4-nitrophenyl-b-D-glucopyranoside; BSA, bovine serum albumin; MALDI-ToF, matrix-assisted laser-desorption
ionization-time of flight; IMAC, immobilized metal affinity chromatography; SPSepharose, sulphopropyl-Sepharose; TLC, thin-layer chromatography; GH3, glycoside
hydrolase family 3; HvExoI, native barley (Hordeum vulgare) b-D-glucan exohydrolase
isoform ExoI; rHvExoI, recombinant barley b-D-glucan exohydrolase isoform ExoI.
91
Recombinant plasmid construction
A full-length HvExoI cDNA from barley seedlings [55] was used as
a template to amplify the native cDNA region encoding the mature
HvExoI (GenBank Accession No. AF102868) with Pfu DNA polymerase and the ExoIMatF (50 -CACCGACTACGTGCTCTACAAGGA-30 ) and
ExoIstopR (50 -CTAGTACTTCTTCGTCGCGTTGGT-30 ) primers. The
exoglucanase I fragment was cloned into the pENTR™/D-TOPO GatewayÒ system entry vector (Invitrogen) according to the manufacturer’s instructions. The cDNA of HvExoI was transferred to the
pET32a/DEST and pPICZaBNH8/DEST vectors by LR clonase recombination reactions (Invitrogen).
The HvExoI cDNA was codon-optimized for expression in P. pastoris and synthesized by GenScript (Piscataway, NJ, USA). The optimized cDNA (GenBank Accession No. GU441535) was cloned into
the pPICZaBNH8 expression vector [53], between the PstI and EcoRI restriction sites by standard methods [56]. The recombinant
bacterial clones were selected on a 25 lg/mL zeocin Lennox broth
(LB) plate, the plasmid DNA isolated and the DNA insert corresponding to HvExoI were sequenced at Macrogen (Seoul, Korea).
Expression of rHvExoI in Escherichia coli
The pET32/DEST expression vector containing the cDNA of
HvExoI was transformed into E. coli strain Origami(DE3) (Novagen,
Madison, WI, USA). Protein was induced with 0.4 mM isopropyl
thiogalactoside (IPTG) at 20 °C for 16–18 h, and extracted as previously described [57]. Protein expression was detected by measuring release of 4-nitrophenol from 4NPGlc. Refolding of rHvExoI
from the insoluble fraction was performed with the iFOLD protein
refolding system 2 kit (Novagen), following the protocol provided
by the manufacturer. In addition, inclusion bodies were washed
with wash buffer containing 0.5% (v/v) TritonX-100, 100 mM NaCl
and 0.1% (w/v) sodium azide in 50 mM Tris–HCl, pH 7.4. Inclusion
bodies were solubilized with the denaturing buffer (6 M guanidine–HCl, 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 10 mM EDTA
and 10 mM DTT) and rHvExoI was refolded by dialysis into the
refolding buffer with gradually decreasing concentrations of guanidine–HCl in 10 mM Tris–HCl, pH 7.4, 10% (v/v) glycerol, 1 mM reduced glutathione and 0.1 mM oxidized glutathione.
Expression of rHvExoI in P. pastoris
The pPICZa-HvExoI plasmids were linearized with PmeI and
transformed into P. pastoris strains Y11430 and SMD11680H (Invitrogen) by electroporation. Colonies were grown on YPDS agar
plates containing 100 lg/mL zeocin, selected again on plates containing 500 lg/mL zeocin and screened for protein production, as
suggested by the manufacturer (Invitrogen).
A single transformed colony was inoculated into 500 mL BMGY
medium (Invitrogen) containing 100 lg/mL zeocin and grown at
28 °C with shaking (160–200 rpm) until the culture reached an
OD600 of 2–3. Cells were harvested by centrifugation (3000g,
5 min, 20 °C) and resuspended in the BMMY medium (Invitrogen)
to reach OD600 of 1. Expression of rHvExoI was induced with 1%
(v/v) methanol for 4 days at 20 °C.
Purification of rHvExoI
The culture broth with secreted rHvExoI was supplemented
with phenyl methyl sulfonyl fluoride (PMSF) to 1 mM and the pH
was adjusted to 4.7 on ice with concentrated acetic acid. The protein solution was loaded onto an SP-Sepharose cation-exchange
column at a flow rate of 1.8 mL/min. The column was washed with
50 mM sodium acetate, pH 4.7, at a flow rate of 0.5 mL/min, and
protein was eluted with a linear gradient of 0–2 M NaCl in
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S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
50 mM sodium acetate, pH 4.7, at a flow rate of 1 mL/min. The active fractions were concentrated and resuspended in 50 mM sodium phosphate, pH 7.8, containing 300 mM NaCl (IMAC buffer).
IMAC purification of rHvExoI was performed by mixing rHvExoI
with Talon Co2+-bound IMAC resin (Clontech, Mountain View, CA,
USA), and then eluting the enzyme with a 0–0.5 M imidazole gradient in the IMAC buffer at a flow rate of 0.5 mL/min. The active
fractions were reconstituted in 20 mM sodium acetate buffer, pH
5.0, by centrifugal filtration (Vivaspin, 10 kDa exclusion limit). Typically, the yield of nearly homogenous rHvExoI (as judged by SDS–
PAGE and immunoblot analyses) was around 4 mg per liter of
culture.
N-deglycosylation of rHvExoI
Purified rHvExoI (60–100 lg) was deglycosylated by 500 U
endoglycosidase H (New England BioLabs, Ipswich, MA, USA) in
50 mM sodium citrate buffer, pH 5.5. The mixture was incubated
at 4 °C for 3–4 days with gentle shaking. Finally, deglycosylated
rHvExoI was purified through a 2nd IMAC column, as described
above, to remove endoglycosidase H. After IMAC, imidazole was removed by centrifugal concentration, as described above and a
nearly homogenous rHvExoI was suspended in 20 mM sodium acetate, pH 5.25.
Tryptic mapping of rHvExoI by MALDI-ToF/ToF spectrometry
About 10 lg of rHvExoI were S-amidomethylated, digested with
100 ng sequencing grade trypsin (Promega, Madison, WI, USA) in
5 mM ammonium bicarbonate and concentrated to 5 ll. A 0.5 ll
aliquot of the digest was applied to a 600 lm AnchorChip (Bruker
Daltonik GmbH, Bremen, Germany) according to the a-cyano-4hydroxycinnamic acid (HCCA, Bruker Daltonik) thin-layer method.
MALDI-ToF mass spectra were acquired with a Bruker Ultraflex III
MALDI-ToF/ToF mass spectrometer (Bruker Daltonik GmbH) operating in reflectron mode under the control of FlexControl, version
3.0 (Bruker Daltonik GmbH). All spectrometry data were processed
with FlexAnalysis, version 3.1 (Bruker Daltonik GmbH), and the
spectra and mass lists were exported to BioTools (Bruker Daltonik
GmbH). The MS and corresponding MS/MS spectra were combined
and submitted to a Mascot database-search.
Molecular mass determination of rHvExoI via MALDI spectrometry
Samples of 1–3 lL of glycosylated and N-deglycosylated rHvExoI were mixed with 1 lL matrix solution (10 mg/mL sinipinic
acid in 90% acetonitrile/0.1% trifluoroacetic acid) and applied to a
600 mm AnchorChip target plate (Bruker Daltonik GmbH, Bremen,
Germany). MALDI-ToF mass spectra were acquired on a Bruker
Ultraflex III MALDI-ToF/ToF mass spectrometer (Bruker Daltonik
GmbH) operating in linear mode under the control of the FlexControl software (Version 3.0, Bruker Daltonik GmbH). External calibration was performed with a mix of ClinProt Protein Calibration
Standard and Protein Calibration Standards 2 (Bruker Daltonik
GmbH), over a range of 1–25 kDa that were analysed under the
same conditions. Spectra were obtained at various locations over
the surface of the matrix spot.
rHvExoI characterization
Enzyme assays and analyses of hydrolytic and transglycosylation activities, substrate specificity, kinetic properties, pH optimum and thermostability were performed as described
previously [38]. The kinetic parameters were determined by a proportional weighted fit, with a nonlinear regression analysis program, based on Michaelis–Menten kinetics [58].
Protein determination, NH2-terminal sequencing, SDS–PAGE and
immunoblot analyses
Protein determination, SDS–PAGE and protein detection on
SDS–PAGE gels were performed as described [34]. Immunoblot
analyses were performed with 0.22 lm nitrocellulose blotting
membranes (Millipore, Billerica, MA, USA), a mouse monoclonal
anti-poly-Histidine-alkaline phosphatase IgG2a isotype antibody
and the BCIP/NBT-purple liquid reagent for membranes, as suggested by the manufacturer (Sigma).
Results
Expression of rHvExoI
A construct in which the HvExoI cDNA was inserted in pET32a/
DEST was first used to express an NH2-terminal thioredoxin-His6tagged fusion protein in E. coli strain Origami(DE3) cells, as this
system has been successfully used for expression of other plant
glycoside hydrolases [54,57,59–61]. Under the conditions tested,
the rHvExoI protein was observed only in the insoluble fraction
upon cell extraction and no enzyme activity was detected (data
not shown). Therefore, refolding of rHvExoI was attempted after
solubilization in guanidine–HCl, but again the enzyme was found
to be inactive under any refolding conditions tested.
Expression of rHvExoI in yeast was tested by inserting native
cDNA into the pPICZaBNH8/DEST expression vector, to produce a
protein fused to the a-factor prepropeptide for secretion. P. pastoris
(Y11430) was tested as a host to produce rHvExoI. No increase in b-Dglucosidase (with 4-nitrophenyl b-D-glucopyranoside, 4NPGlc) or
exoglucanase (with barley (1,3;1,4)-b-D-glucan) activities were observed and the protein could not be detected on Coomassie-stained
SDS–PAGE gels in the predicted mass range (data not shown). Since
this construct contains an eight-histidine tag at the NH2-terminus of
rHvExoI (AHHHHHHHHAA) after the secretory peptide cleavage site,
the rHvExoI could be detected by immunoblot analysis with antipolyhistidine antibody. Here, a 43 kDa band was detected, which
could be correlated to the size of domain 1 of rHvExoI (data not
shown). This analysis indicated that either rHvExoI was produced
as a full-length protein and then proteolytically degraded by an
unspecified protease from the host, or that premature termination
of translation occurred near the end of domain 1, possibly because
the native barley cDNA contained codons of low usage in P. pastoris.
To avoid the problems of proteolysis and poor codon usage for protein synthesis in P. pastoris, the HvExoI cDNA was codon-optimized
and expressed in the protease-deficient P. pastoris strain SMD1168H.
The native and codon-optimized rHvExoI cDNA fusions were
transformed into P. pastoris strain SMD1168H and protein expression was induced at a temperature of 20 °C. High levels of HvExoI
activity, measured with 4NPGlc, were detected from the codonoptimized rHvExoI cDNA fusion and a series of bands from 75 to
85 kDa were detected by immunoblot analysis with anti-polyhistidine antibody (Fig. 1A). The activity in the media slowly increased
until 4 days of induction, after which it did not change. However,
little increase in rHvExoI activity and protein production (as determined by SDS–PAGE and Coomassie-staining) were observed from
the cDNA fusions containing codon-optimized HvExoI that were
induced at a temperature of 28 °C, or from a construct containing
the native HvExoI cDNA induced at any temperature (data not
shown).
rHvExoI purification
After rHvExoI was expressed in P. pastoris from the codon-optimized cDNA at 20 °C for 4 days, a set of major protein bands, with
S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
93
Table 2
Amino acid sequences of tryptic fragments of rHvExoI identified by MALDI-ToF/ToF.
Tryptic fragmentsa
1
2
3
4
5
6
7
8
MTLAEKIGQMTQIERLVATPDVLRDNFIGSLLSGGGSVPR
GATAKEWQDMVDGFQK
RIGEATALEVRATGIQYAFAPCIAVCR
RIVQSMTELIPGLQGDVPKDFTS GMPFVAGK
HFVGDGGTVDGINENNTIINREGLMNIHMPAYKNAMDKGVSTVMISYS
SWNGVKMHANQDLVTGYLKDTLKFKGFVISDWEGIDRITTPAGSDYSYS
VKASILAGLDMIMVPNK
RVKFTMGLFENPYADPAMAEQLGK
NGKTSTDAPLLPLPK
TTVGTTILEAVKAAVDPSTVVVFAENPDA
EFVKSGGFSYAIVAVGEHPYTETKGDNLNLTIPEPGLSTVQAVCGGVR
9
a
Fig. 1. Protein production of rHvExoI detected by immunoblot analysis (A) and
SDS–PAGE (B). (A) Expression of rHvExoI in P. pastoris was detected via anti-His-tag
antibodies by immunoblot analysis. Mr is a prestained protein marker, lanes 1–3
indicate levels of rHvExoI production after 1, 2 and 3 days, respectively. (B) SDS–
PAGE of rHvExoI from crude broth (lane 1), and purified via SP-Sepharose
chromatography (lane 2), the 1st IMAC step (lane 3), and the 2nd IMAC step, after
deglycosylation with endoglycosidase H (lane 4).
apparent masses corresponding to the molecular mass of rHvExoI
and higher, was detected by immunoblot analysis and SDS–PAGE
with Coomassie staining (Fig. 1B). This protein was purified from
the culture media by SP-Sepharose chromatography (Table 1). Protein was eluted with 1.2 M NaCl in 50 mM NaOAc, pH 4.7. The fractions containing narrow ranges of activity near the Gaussian peak
(based on activity, absorbance at 280 and SDS–PAGE profiles) were
concentrated and purified by Immobilized Metal Affinity Chromatography (IMAC), which bound rHvExoI containing the His8-tag. A
small amount of exoglucanase activity, which might have originated from the presence of rHvExoI without a His8-tag, passed
through the column. A portion of the activity that passed through
the column may also be attributed to a native P. pastoris (1,3)-b-Dglucan exohydrolase [62]. Nevertheless, most of the expressed
rHvExoI was eluted from the column with 250 mM imidazole
and the specific activity of rHvExoI was increased by about 4-fold
after IMAC purification (Table 1). It has been reported that native
HvExoI has three N-glycosylation sites at Asn221, Asn498 and
Asn600 [39]. To test whether the bands detected in the range between 80 and 100 kDa (Fig. 1B) were due to the presence of
over-N-glycosylated forms of rHvExoI, and whether these N-glycosylation sites could affect enzyme properties, the N-linked carbohydrate was removed by endoglycosidase H. After Ndeglycosylated rHvExoI was purified by a second round of IMAC,
the specific activity remained more-or-less unchanged (Table 1).
The amino acid sequence of N-deglycosylated rHvExoI was confirmed by mass spectrometry (Table 2; vide infra).
Table 1
Enzyme yields during purification of rHvExoI.
Purification
step
Yielda
Protein
(mg)
Crude
119
protein
SP18.9
Sepharose
1st IMAC
3.3
2st IMAC
2.9
a
b
c
d
Specific
activitya
(units mg
Recoverya,c Purification
(%)
factora,d
1
)
(fold)
Activityb
(units)
SVDQLPMNVGDAHYDPLFRLGYGLTTNATK
The underlined letters indicate N-glycosylated Asn (N) residues.
Tryptic mapping of rHvExoI
Purified N-deglycosylated rHvExoI (Fig. 1B) was digested with
trypsin, and the molecular masses of individual tryptic fragments
were determined by MALDI-ToF/ToF spectrometry, which provided
a coverage of 65% of the ions with expected m/z values. Nine peptide sequences (Table 2) deduced from the nucleotide sequences of
the pPICZa-HvExoI DNA fusion had predicted m/z values that exactly matched those ions that were observed during mass spectrometry analyses. The three peptides containing the putative Nlinked glycosylation sites had masses that were 203.2 Da higher
than those of the predicted peptides. The additional mass of
203.2 Da corresponded to a single N-acetyl b-D-glucosaminyl residue, which remained attached to the asparaginyl residues after the
remainder of the sugar had been cleaved off by endoglycosidase H.
Molecular mass and NH2-terminal sequencing of rHvExoI
The full-length, His-tagged rHvExoI of 67.1 kDa was observed in
the zip-tip purified N-deglycosylated sample by MALDI-ToF mass
spectrometry (Fig. 2A). A doubly charged form of rHvExoI was also
observed at 33.6 kDa and a very low abundance form of 44.7 kDa
was also seen, along with a likely doubly charged form of this species at 22.5 kDa. However, the MALDI-ToF spectra obtained from
the protein sample that had not been N-deglycosylated were undefined and did not provide clear mass indications. This N-glycosylated protein sample, which was expected to contain multiple Nglycosylated forms of rHvExoI with different molecular masses, resulted in the very low intensity spectra shown in Fig. 2B. Subsequent analyses of these two samples by an electrospray
ionisation ion trap mass spectrometry failed to detect any masses
above the background ions, therefore NH2-terminal protein
sequencing of N-deglycosylated rHvExoI was undertaken. The data
revealed that the AHHH amino acid sequence was indeed present
in rHvExoI and that the EAEA signal cleavage sequences had been
removed from the NH2-terminal region of the otherwise full-length
rHvExoI enzyme [63].
Effect of pH and temperature on enzyme activity
98.7
0.8
100
41.3
2.2
41.9
2.6
11.7
9.0
3.5
3.1
11.8
9.1
4.2
3.7
The numbers were rounded to the nearest decimal place.
Recovered enzyme units assayed on 4NPGlc.
Expressed in percent of enzyme activity in the crude extract.
Calculated on the basis of specific activity (units/mg protein).
1
The pH optima of the glycosylated and N-deglycosylated forms
of rHvExoI were determined in McIlvaine buffer over the pH range
of 3.5–8.5 at 30 °C (Fig. 3). The pH profiles were bell-shaped, and
the highest activities of glycosylated and N-deglycosylated rHvExoI
were detected at pH 5.0, although the pH profile suggested the
optimum for the N-deglycosylated rHvExoI should be 5.25
(Fig. 3). As for the temperature stability of rHvExoI, the activities
were assayed after incubation at temperatures over the range between 0 and 80 °C for 15 min (Fig. 4). While glycosylated rHvExoI
94
S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
Fig. 2. MALDI-ToF spectra of N-deglycosylated (A) and glycosylated (B) rHvExoI.
was stable at the temperature range between 10 and 20 °C, Ndeglycosylated rHvExoI was stable in the range between 10 and
30 °C. Above these temperature ranges, the activities of both rHvExoI forms decreased substantially (Fig. 4). The temperatures at
which 50% inactivation of the glycosylated and N-deglycosylated
rHvExoI forms were observed, were 34 and 39 °C, respectively.
Both rHvExoI forms were denatured at 50 °C and no activity was
detected at higher temperatures (Fig. 4). The addition of BSA at
160 mg/L could marginally stabilise and protect the rHvExoI protein against heat inactivation (data not shown).
Substrate specificity
Fig. 3. pH-activity profile of glycosylated (- -) and N-deglycosylated (—) rHvExoI.
Activity was assayed with 0.2% (w/v) 4NPGlc, 160 lg/mL BSA in 0.1 M citric acid–
0.2 M disodium hydrogen phosphate (McIlvaine buffers) over the pH range of 3.5–
8.5.
The substrate specificities of the glycosylated and N-deglycosylated rHvExoI enzymes were determined towards the polysaccharides laminarin, barley (1,3;1,4)-b-D-glucan and lichenan and the
synthetic glycoside 4NPGlc at 0.2% (w/v) concentrations. The data
indicated that rHvExoI was similar to native HvExoI, in that it
hydrolysed polysaccharides, including barley (1,3;1,4)-b-D-glucan
and laminarin and lichenans from Icelandic moss (Cetraria islandica),
as well as the aryl-b-D-glucoside substrate 4NPGlc (Table 3). The
rHvExoI enzyme was specific for glucosides and could not hydrolyse a-L-arabinoside, b-D-galactoside, b-D-xyloside, and b-D-fucoside (data not shown). Comparative analyses of the activities of
glycosylated and N-deglycosylated rHvExoI indicated that both enzymes behaved similarly with laminarin and barley (1,3;1,4)-b-Dglucan, although glycosylated rHvExoI had higher activity with
4NPGlc than the N-deglycosylated form of rHvExoI (Table 3).
Kinetic and inhibition parameters
Fig. 4. Thermostability of glycosylated (- -) and N-deglycosylated (—) rHvExoI.
rHvExoI was assayed with 4NPGlc for 15 min at 30 °C after being incubated at the
indicated temperatures between 0 and 80 °C for 15 min.
The kinetic parameters for hydrolysis of laminarin, barley
(1,3;1,4)-b-D-glucan, and 4NPGlc were determined for both glycosylated and N-deglycosylated rHvExoI (Table 4). Both forms were
highly active on polysaccharides and the most efficient polymeric
substrate was barley (1,3;1,4)-b-D-glucan, which was hydrolysed
approximately 1.5- to 2-fold more efficiently, in terms of the
kcat/KM catalytic efficiency values than laminarin. The hydrolysis
of barley (1,3;1,4)-b-D-glucan proceeded about 1.3-fold faster with
S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
95
Table 3
Relative activities of rHvExoI on polysaccharides, oligosaccharides and synthetic
substrates.
Relative activity (%)a
Substrate
Native
HvExoIb
Glycosylated
rHvExoI
N-deglycosylated
rHvExoI
Polysaccharides
Laminarin (L. digitata)
Barley (1,3;1,4)-b-D-glucan
Lichenan (C. islandica)
100c
10
nme
98.9 ± 1.1d
10.0 ± 0.5
1.37 ± 0.09
98.8 ± 1.2d
10.0 ± 0.8
nm
Synthetic substrate
4-NPGlc
10
22.8 ± 1.4
16.8 ± 0.8
a
The numbers were rounded to three significant figures.
b
The data are from [38].
c
The relative activity of 100% equals to 63 U/mg.
d
The relative activity of laminarin equals to 54.4 and 65.3 U/mg of glycosylated
and N-deglycosylated forms of rHvExoI, respectively.
e
‘nm’ indicates ‘not measured’.
Table 4
Kinetic parameters of rHvExoI on laminarin, barley (1,3;1,4)-b-D-glucan, and 4NPGlc.
Substrate
Native HvExoIa
Laminarin
KM (mM)
kcat (s 1)
kcat/KM (mM
0.098b
73b
740b
1
s
1
)
Barley (1,3;1,4)-b-D-glucan
0.012b
KM (mM)
4b
kcat (s 1)
330b
kcat/KM (mM 1 s 1)
4NPGlc
KM (mM)
kcat (s 1)
kcat/KM (mM
1
s
1
)
1.4b
5b
3b
Glycosylated
rHvExoIa
N-deglycosylated
rHvExoIa
0.18 ± 0.01
42.1 ± 1.6
234 ± 14
0.22 ± 0.02
52 ± 3
240 ± 20
0.044 ± 0.004
14.1 ± 0.7
365 ± 57
0.020 ± 0.002
9.7 ± 1.1
478 ± 50
1.99 ± 0.15
27.7 ± 0.9
13.9 ± 0.6
2.0 ± 0.12
25.4 ± 0.7
12.8 ± 0.8
Fig. 5. TLC chromatogram of hydrolysis and transglycosylation products formed by
glycosylated rHvExoI. The enzyme was incubated in the presence of 20 mM 4NPGlc
at 30 °C for 0, 3 min, 4 h and 18 h. Standards are glucose (Glc), laminarioligosaccharides (L2–L7), cello-oligosaccharides (C2–C6), and gentiobiose (Gen),
4NP-b-laminaribiose (4NPLam), and 4NP-b-cellobiose (4NPCel). The presence of
oligosaccharide products with unknown structures are indicated as P1–P4. The
reaction times are indicated in hours below the lanes.
a
The values were rounded to three significant figures, or to the precision of the
error.
b
Kinetic parameters of HvExoI with laminarin, barley (1,3;1,4)-b-D-glucan and
4NPGlc are from [38].
the N-deglycosylated rHvExoI form, although with the other two
substrates, the hydrolytic rates were similar (Table 4). The kcat/
KM catalytic efficiency values of rHvExoI were higher than those
of native HvExoI for barley (1,3;1,4)-b-D-glucan and 4NPGlc, while
native HvExoI was more efficient at hydrolysing laminarin
(Table 4).
Transglycosylation
Both the glycosylated and N-deglycosylated forms of rHvExoI
possessed both hydrolytic and glucosyltransferase activities toward 4NPGlc at 20 mM concentration. Here, the formation of
transglycosylation products was detectable within 3 min of reaction (0.05 h; Fig. 5). The transglycosylation product patterns of Ndeglycosylated rHvExoI were very similar to those of glycosylated
rHvExoI shown in Fig. 5. During the initial stages of the reaction at
3 min, a small amount of glucose was observed, in addition to 4NPgentiobioside and 4NP-laminaribioside. The 4NP-oligosaccharide
products were identified by comparison with standard compounds
of known Rf values [64]. As the reaction progressed, after 4 h reaction, the transient transglycosylation products laminaribiose, cellobiose, 4NP-laminaribioside, 4NP-cellobioside and an unknown
disaccharide (P1) and trisaccharides (P2–P4) were detected. By
18 h, the 4NP-glycosides, laminaribiose and cellobiose were hydrolysed, and only the major hydrolytic product glucose and traces of
gentiobiose were observable (Fig. 5). Transglycosylation activity
also occurred with barley (1,3;1,4)-b-D-glucan, from which only
the formation of the disaccharide gentiobiose was detected (data
not shown). Similar observations were reported for native HvExoI
[38].
Discussion
Development of an expression system for active rHvExoI
Bacteria and yeast are common hosts for production of target
proteins. Although, bacterial expression is convenient, many
eukaryotic proteins are produced in low protein yields [65,66] or
are produced as insoluble aggregates [67–69]. Although HvExoI
could be expressed in bacteria, it was not able to fold properly,
and refolding experiments using various refolding formulations
failed to recover active rHvExoI. The reasons why rHvExoI could
not be refolded successfully might include its two-domain organisation and the fact that the enzyme contains 10 cysteine residues.
From these residues, two disulfide linkages are formed, between
Cys151 and C159 in domain 1, and between Cys513 and Cys518
in domain 2 [39]. Hence, the probability that the correct disulfide
linkages will be formed is low. Therefore, a yeast expression system was introduced that was reported to be able to produce plant
enzymes in active forms (e.g. [63]).
Prokaryotic and eukaryotic cells have their own species-specific
codon usage patterns. Many target genes from mammals and
plants are expressed at low levels in bacteria or yeast, in part because the rate of protein translation is not well correlated to codon
usage and tRNA bias [70]. The HvExoI cDNA amplified from barley
96
S. Luang et al. / Protein Expression and Purification 73 (2010) 90–98
seedlings produced low protein yields in P. pastoris. Thus, a codonoptimized cDNA was synthesised. Expression of rHvExoI was only
successful from the codon-optimized cDNA in a protease-deficient
strain of P. pastoris and at a low temperature of 20 °C. The expressed rHvExoI possessed three post-translationally modified
asparaginyl residues, similar to native HvExoI. rHvExoI could be
purified by a two-step procedure using a strong cation-exchanger
and IMAC with a final yield of 12%, based on the total protein used
for purification. The use of selective pooling of the chromatographic fractions, as well as initial contamination with P. pastoris
exoglucanase [62], which was eliminated during the purification,
and loss of a small amount of HvExoI that lacked its N-terminal histidine tag may account for the relatively lower apparent final yield
of purified enzyme that was around 10–12%. Upon N-deglycosylation with endoglycosidase H and purification with a second IMAC
step, N-deglycosylated rHvExoI was produced with a yield of 9%,
and the specific activity of rHvExoI was nearly the same as before
N-deglycosylation. Here, the endoglycosidase H was chosen, because it cleaves the chitobiose core of high mannose N-linked glycoproteins, which are commonly observed in yeast, while retaining
the first N-acetylglucosamine residue linked to Asn [71]. A Histagged form of rHvExoI of 67.2 kDa lacking an EAEA secretion motif
was obtained after expression in P. pastoris and N-deglycosylation.
Thus, secreted rHvExoI was properly processed by the proteases in
Pichia cells, contrary to previous observations with xyloglucan
xyloglucosyl transferase enzymes [63].
Catalytic properties of rHvExoI
The recombinant glycosylated and N-deglycosylated rHvExoI
forms were unique in terms of their pH-activity profiles in the
range of pH 3.5–8.5 and thermostabilities. The pH optimum and
thermostability of N-deglycosylated rHvExoI were similar to native
HvExoI, although the same parameters of glycosylated rHvExoI
were slightly lower. Koseki et al. [72] reported that the N-glycosylation of oligosaccharide chain (2.5 kDa) of asparagines at the catalytic domain increased the thermostability of Aspergillus kawachii
a-L-arabinosidase, whereas glycosylated rHvExoI did not exhibit
increased stability at elevated temperatures. The molecular mass
of glycosylated rHvExoI was higher by approximately 7.7–
17.7 kDa than that of N-deglycosylated enzyme due to occupation
of three N-glycosylation sites. The calculated molecular mass of
HvExoI was 66.8 kDa [37]. Based on the sizes of N-linked carbohydrates at the N-glycosylation sites, we would expect that the presence of these carbohydrates could affect certain enzyme
properties, such as pH optimum and thermostability. However,
the glycosylated and N-deglycosylated rHvExoI enzymes had pH
optima and thermostabilities quite similar to native HvExoI [38].
The polysaccharides laminarin and barley (1,3;1,4)-b-D-glucan
and the aryl glycoside 4NPGlc were hydrolysed by rHvExoI with
similar hydrolytic rates, compared to native HvExoI. It has well
been documented that native HvExoI prefers hydrolysing substrates containing (1,3)-b-linked glucoside residues [37,38,42]. As
for the catalytic properties, we found that rHvExoI had kcat/KM values very similar to native HvExoI with barley (1,3;1,4)-b-D-glucan,
although the kcat/KM value of rHvExoI was higher with 4NPGlc, and
about 3-fold lower with laminarin (Table 4). The only obvious differences between the rHvExoI and native HvExoI enzymes is the
presence of the extension at the N-terminus by 11 residues
(AHHHHHHHHAA) in rHvExoI, and differences in N-glycosylation
of both forms produced in P. pastoris. The 11-residue extension
and the Asn600 glycosylation site lie at the N- and C-termini of
HvExoI, respectively, distant from the active site, however, the
Asn221 and Asn498 N-linked glycosylation sites lie in the proximity of the active site of HvExoI [39,40]. It is possible that the native
barley carbohydrate might help in interaction with a polymeric
substrate, such as laminarin, resulting in the much tighter binding
seen in the native enzyme. However, it appears the high mannose
and N-acetyl glucosamine containing carbohydrates affixed to
these sites by P. pastoris makes no significant net interaction with
substrates. It is also possible that the differences in N-terminal sequence and posttranslational modification result in differences in
protein flexibility or other protein physical properties that might
explain the differences in hydrolysis of these substrates.
Conclusions
Recombinant rHvExoI was expressed at high levels from a codon-optimized HvExoI cDNA in protease-deficient P. pastoris, under low temperature conditions, while the expressions from a
native HvExoI cDNA in E. coli or P. pastoris were unsuccessful. rHvExoI exhibited hydrolase activities with b-linked polysaccharide
and aryl glucoside substrates and produced a variety b-linked oligosaccharide products through transglucosylation activities, similar to the native HvExoI enzyme. Thus, the recombinant rHvExoI
is an appropriate model enzyme to study the roles of amino acid
residues in catalysis and substrate specificity, as revealed by
numerous crystal structures of HvExoI, by site-directed
mutagenesis.
Acknowledgments
We are grateful to Dr. Kris Ford (University of Melbourne, Australia), Mark Condina and Alexander Colella (Adelaide Proteomics
Centre, the University of Adelaide, Australia) for mass spectrometry, molecular mass and sequencing analyses. Tassanee Onkoksoong is gratefully acknowledged for generation of the
pPICZaBNH8/DEST plasmid. This work was supported by grants
from the Australian Research Council to MH, the Thailand Research
Fund (TRF) Grant BRG5080007 to J.R.K.C. and a TRF Royal Golden
Jubilee Ph.D. Fellowship to S.L.
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