Construction of a Vector for the Surface Display of Heterologous Proteins in Saccharomyces cerevisiae
An E. coli yeast shuttle vector for the anchoring of heterologous protein to the yeast hosts cell wall was constructed. The vector PYDSM01 includes a DNA sequence constructed from the signal sequence from the yeast sucrose isomerase gene, a multiple cloning site and a DNA fragment encoding the carboxyl-terminal of the yeast cell wall protein 2 (CWP2). This construct was then inserted into the HindIII site on pGAD424, replacing the GAL4 fusion tag and the original MCS sequence. DNA sequencing confirmed the correct insertion of both signal and anchor proteins in the vector. To test for proper expression and functional anchoring to the cell wall, the coding sequence for a bacterial alpha-amylase enzyme was cloned into the vector and transformed into a yeast host. A total of 22 yeast transformant were recovered which were able to degrade starch, indicating successful expression and function of the bacterial alpha-amylase gene. Enzyme assay of the washed cell pellet and supernatant fractions indicate that the both activity and anchoring efficiency are variable.
Received: August 04, 2011;
Accepted: November 23, 2011;
Published: January 14, 2012
The use of the yeast Saccharomyces cerevisiae to display heterologous-expressed
proteins on the cell surface was pioneered two decades ago (Schreuder
et al., 1993). In the early phases, yeast surface display was used
mainly for immobilization of enzymes (Pepper et al.,
2008). A variety of enzymes such as amylase and glucoamylase (Schreuder
et al.,1993; Murai et al., 1997a),
cellulolytic enzymes (Murai et al., 1997b; Murai
et al., 1998; Fujita et al., 2002)
and lipase (Matsumoto et al., 2004) has been
successfully displayed on the yeast cell wall. More recently, several groups
have successfully demonstrated the simultaneous anchoring of multiple enymes
e.g., β-glucosidase and carboxymethylcellulase (Murai
et al., 1998) and also glucoamylase and α-amylase of S. diastaticus
and B. amyloliquefaciens origin respectively in the same yeast cell
(Steyn and Pretorius, 1991). Co-expression of glucoamylase
of Rhizopus oryzae and α-amylase of B. stearothermophilus
(Murai et al., 1999) were further found to enhance
the stability of both enzymes. Surface display permits more than single modification
to be carried-out on the subject protein and some has reported a synergistic
combination of protein engineering and display system e.g., when lipase of R.
oryzae origin was displayed on cell surface of S. scerevisiae in
organic solvents, the catalytic activity was several folds higher than the original
construct (Shiraga et al., 2005). Functional
minicellulosome complexes consisting of endoglucanase, cellobiohydrolase and
β-glucosidase arranged on a scaffold molecule has been expressed and assembled
on the surface of yeast, enabling the simultaneous breakdown and fermentation
of cellulose to ethanol (Pepper et al., 2008).
Besides enzymes, pioneering work by Boder and Wittrup (1997)
used yeast surface display to create combinatorial protein libraries of human
antibody Fab fragments for affinity selection. This was further developed to
include application for other human proteins with therapeutic potential e.g.,
T-cell receptors (Pepper et al., 2008). Other
applications include the displaying of fluorescent protein as a reporter gene
(Shibasaki et al., 2001), protein A (SpA) for
immunoglobulin binding (Nakamura et al., 2001),
a histidine oligopeptide (Hexa-His) for chelating heavy metal ions (Kuroda
and Ueda, 2003; Kuroda et al., 2001) and
the endocrine disruptor group of proteins (Routledge and
Sumpter, 1997) for environmental screening of waste water.
Early methods for anchoring proteins onto the yeasts cell wall were achieved
by using either the β-agglutinin Aga2p or flocculin Flo1p as the cell wall
anchor protein. The Aga2p system, developed by Boder and
Wittrup (1997) anchors the fusion protein by disulfide bonding to Aga1p
on the yeasts cell wall. The Flo1p protein is used when non-covalent display
is desired (Kondo and Ueda, 2004). The Pir family of
cell wall proteins was also used to display human glycosyltransferase enzymes
(Shimma et al., 2006). The selection of a suitable
anchor appears to play an important role in successful expression and functionality
of the fusion protein (Abe et al., 2003). Earlier
work by Van der Vaart et al. (1997) compared
the effectiveness if several yeast cell wall proteins Cwp1p, Cwp2p, Aga1p, Tip1p,
Flo1p, Sed1p, YCR89W and Tir1p in functionally displaying an β-galactosidase
enzyme and concluded that a the C-terminal fragment of Cwp2p was the most effective
anchor. Breinig and Schmitt (2002) used Cwp2p to express
the haemagglutinin (HA) epitope, and subsequently a bacterial esterase (Breinig
et al., 2006). Besides these, however, Cwp2p was not widely used
as an anchor.
While the technology for yeast surface display have been taken to a new level of sophistication, there is still a need for a simple system capable of displaying proteins of industrial interest e.g., enzymes at high density, while being firmly bonded to the cell. The most widely used Aga2p system relies on disulfide bonds between Aga1p and Aga2p for attachment. For this purpose, we design a vector for yeast surface expression using the 67 C-terminal amino acids of Cwp2p as an anchor.
MATERIALS AND METHODS
Construction of pYDSM01: The backbone of surface-display vector was
derived from plasmid pGAD424, a yeast two-hybrid vector (Clontech, USA). The
GAL4 AD domain fusion tag and Multiple Cloning Site (MCS) of pGAD424 was excised
by digestion with HindIII. This resulted in a linearised plasmid backbone
with HindIII overhangs. The two fragments were separated on a 0.8% agarose
gel by electrophoresis and the backbone recovered by band excision and extraction
using a commercial kit (Qiagen, USA). The linearised plasmid was then dephosphorylated
using Antartic phosphatase (NEB).
A two-step PCR strategy was used to build the cloning construct (Fig.
1). Primers YS1F 5- ATCGAGAATTCCCGGGGATCCGTCGACCTGCAGAGATCTATATTTCTCAAATCACTGAC
GGTC and YS2R 5- ATCGAAAGCTTTTATAACAACATAGCAGCAGCAG were used to
amplify a fragment coding for 67 amino acids on the C-terminal of CWP2.
The first 30 nucleotides of the forward primer YS1F contain part of the signal
sequence for the yeast sucrose isomerase and a Multiple Cloning Site (MCS) sequence.
Yeast genomic DNA was extracted using QIAGENs Dneasy kit and used as the
||Schematic diagram for vector construction (Wahab
et al., 2011). (SucSg = Signal sequence of sucrose isomerase)
The required fragment was generated by Polymerase Chain Reaction (PCR) with
a denaturation temperature of 94°C for 15 sec, annealing at 56°C for
30 sec, extension at 68°C for 1 min, for 30 cycles on an Eppendorf Mastercycler.
The resulting amplification product from this amplification step contains part
of the 3 end of the sucrose isomerase signal sequence, an in-frame MCS
and sequences coding for the N-terminal 67 amino acids of Cwp2p.
A second set of primers was used to make the complete insert. The forward primer (YS3F) consists of the first 40 nucleotides of the sucrose isomerase signal sequence. The internal HindIII site in the signal sequence was abolished by site-directed mutagenesis by substituting A for T in the fifth codon. The resulting mutation is silent. A flanking HindII site was added immediately before the start codon. The reverse primer was YS2R. The PCR product was purified and verified by sequencing. The fragment was then digested with HindIII and ligated into the linearised pGAD424 backbone. Post ligation, the reaction mix was transformed into E.coli DH5 cells. Screening was carried-out by plating on Luria-Bertani (LB) agar plates supplemented with ampicillin (100 mg L-1). The resulting vector (PYDSM01) was recovered from positive transformants and verified by restriction enzyme mapping and DNA sequencing.
Cloning of the bacterial α-amylase gene: Genomic DNA was extracted
from Bacillus subtilis ATCC 6633 using Qiagens DNeasy kit and used
as the source of bacterial amylase gene. PCR was carried out using primers BA1F
and BA2R targeting the entire ORF minus the start and stop codons. PCR was performed
at 94°C for 15 sec, annealing at 55°C for 30 sec, extension at 68°C
for 3 min, for 30 cycles on an Eppendorf Mastercycler. The amplification product
was purified using a commercial kit and sequenced for verification. The purified
PCR product was digested with BamHI and cloned into pYDSM01 at the BamHI
site in the MCS. Post ligation, the reaction mix was transformed into E.coli
DH5α cells as described above. Plasmids were recovered from positive
transformants and the insertion was verified by sequencing. For expression and
surface display, the recombinant plasmid was transformed into a leu2 yeast strain
using the Lithium acetate method of Gietz (1992). Putative
transformants were selected on synthetic complete media lacking leucine and
Cloning the GFP gene: The coding sequences for a variant of the green fluorescent protein optimized for expression in yeast was amplified from plasmid pFA6a-GFP-KanMX (a gift from R.Borts, U.Leicester). The amplified sequence were purified, sequenced, digested with BamHI and inserted into pYDSM01 as described above. Transformants were selected on synthetic complete media lacking leucine and characterized.
Characterization of yeast transformants: Yeast transformants were cultured individually in 96-well plates in 100 μL of YEPD. After overnight growth at 30°C, 100 μL of 1% starch solution were added to each well and incubated for 1 h at 30°C. A drop of Grams iodine was added to detect for residual starch. Transformants with amylase activities were selected, inoculated into YEPD supplemented with 100 mg L-1 ampicillin to prevent bacterial contamination and grown overnight at 30°C. The cells were then recovered by centrifugation, washed twice and resuspended in phosphate buffer (pH 7). Both the washed cells and the culture supernatant were then assayed for amylase activity by testing for the production of free glucose from starch using the AMPLEX Glucose kit (Invitrogen, USA). Amylase activity was measured in three replicates for each transformant and the results averaged.
For transformants carrying the GFP insert, cells grown overnight in YEPD were harvested by centrifugation, washed twice and resuspended in phosphate buffer. An aliquot was mounted in antifade mountant (Invitrogen) and observed under a fluorescent microscope using appropriate filters. A control consisting of yeast cell transformed with the vector only was prepared and treated in exactly the same manner.
RESULTS AND DISCUSSION
Vector construction and gene cloning: Successful construction of vector
PYDSM01 was verified by sequencing (Fig. 2). DNA sequencing
confirms the correct orientation and reading frame of the insert.
||DNA sequence and corresponding amino acid sequence of the
construct. The first 20 codons codes for the sucrose isomerase signal sequence.
The asterisk *marks the nucleotide that was mutated to abolish HindIII
restriction site. Sequences in small letters indicate part of the engineered
MCS, followed part of the 5 end of the B.subtilis alpha-amylase
gene. The entire construct was sequenced and verified to be free of PCR-induced
mutations. The sequences were also checked at both BamHI junctions
(italics and underlined) to ascertain that the entire construct is in-frame
with respect to all the three sequences. The first 6 codons of the CWP2
fragment are shown in the figure (underlined)
||Screening for yeast transformants displaying amylase activity.
Wells A1 to G1 contain yeast cells transformed with pGAD424 as controls.
No amylase ac tivities were observed in these cells, as evident by the dark
blue color of the starch-iodine complex. Clear wells shows reactions where
the starch has been hydrolysed, indicating transformants with amylase activity
Preliminary screening of transformants: Yeast transformants were first selected for the presence of amylase activity using a simple starch hydrolysis assay. Figure 3 shows an example of a 96-well microtiter plate screening of transformants expressing the α-amylase enzyme. The yeast host that is used does not contain an amylase gene and no amylase activity was observed in the non-transformed yeast cells (data not shown) or cells transformed with the vector only. Clear, or very light purple wells can be seen at A3,A7, B3- 4, B8, C2, C4- 5, C8, D2- 3, D6- D8, E3, F3 and G3-4. All transformants which tested positive for starch hydrolysis were selected for secondary screening. While the expression of the amylase gene appears to be a stable phenotype, the level of expression differs among the positive transformants (Fig. 4). Some transformants were able to completely hydrolyse the starch substrate within 30 min (clear wells) while in some wells a light purple color, indicative of residual starch, can still be observed at the end of the incubation period.
Glucose assay: Table 1 shows results from glucose assay of the amylase activity of the cell and supernatant fractions from selected transformants. The transformants were grown in YEPD and centrifugation was used to separate the cell and supernatant. The cell pellet was washed and resuspended in PBS.
Sample Y0 was the original yeast host transformed with the unmodified pGAD424
vector. No amylase activity was detected in either the cell or supernatant fractions.
For transformants Y14 and Y22, amylase activity was detected only in the supernatant
fractions. These represent transformants in which the amylase gene was successfully
expressed and secreted out of the cell, but the fusion protein was not incorporated
into the yeast cell wall. Possible explanation includes premature truncations
of protein synthesis, leading to the loss of the Cwp2p tag. For transformants
Y5, Y7 and Y9, the amylase activity was detected only on the cell pellet. These
represent transformants in which the recombinant construct was successfully
expressed, secreted and anchored correctly on the cell wall. Except for transfrormant
Y5, the cell pellet fractions in general display lower enzyme activities compared
to the supernatant.
||Secondary screening for yeast transformants that express amylase
activities. Column A1 to E4 are cells transformed with the vector only.
The expression of the alpha-amylase gene appears to be variable among positive
||Yeast cells transform with vector and GFP insert fluorescing
green color when excited with blue light. In (a) the fluorescence is concentrated
near the cell surface. (b) Fluorescence appears to be diffused in the cell
It is possible that anchoring by Cwp2p somehow interferes with the amylase
In six transformants (Y2, C1-C5), substantial amylase activities was found
on both the cell pellet and supernatant fractions. These are transformants in
which the construct was successfully expressed at a high level but a portion
of the amylase enzymes were not properly anchored. Similar observations were
reported by Murai et al. (1997a) with fungal
amylases and were hypothesized to be caused by proteolytic processing of the
fusion protein, leading to loss of the anchor tag. Transformants C1-C5 was obtained
from a different batch of experiment. It appears that the physiology of the
host cell during transformation may have an effect on the processing of the
No activity was detected from transformant Y18 which showed positive results in the preliminary screening. This was also observed in a number of transformants that lost their amylase activity during the secondary screen.
||Detection of amylase activity on the cell pellet and supernatant
fractions of transformants. Y0 represent the average reading for more than
10 clones of yeast cells transformed with the vector only
|ND: Not detected
The recovery of three types of transformants indicate that the constructed vector pYDSM01 is able to expressed a cloned gene fused to a signal sequence and a C-terminal Cwp2p tag that anchors to the cell wall of a yeast host. However, expression appears to be variable in some transformants. Some transformants also failed to have the fusion protein displayed on the cell wall, although enzyme activity was detected, indicating successful expression but not anchoring. This variable expression and anchoring can be due to a number of factors e.g., instability of the recombinant plasmid, variable promoter activity or premature truncation of the fusion protein resulting in loss of enzyme activity or the Cwp2 tag.
It is also possible that we tried to express a bacterial enzyme, which may
not be fully functional in the yeast host cell. For prokaryotic enzymes, proper
folding appears to be required for maximum activity (Shimma
et al., 2006).
Expression of GFP: Yeast cell transformed with the vector containing cloned GFP sequences were observed to fluoresce after overnight growth (Fig. 5). In a number of cells, the fluorescence pattern appears to be localized to the periphery of the cell, indicating that the GFP is concentrated on the cell wall. In some cells however, the green fluorescence appears to be diffuse within the cell, possibly indicating free proteins in the cytoplasm. There were also non-fluorescing cell. No fluorescence was observed in the negative control which are transformants carrying an empty vector. All samples and controls were processed in the same manner after the same period of growth.
The same variable phenotypes were thus observed for GFP as in yeast transformants
expressing amylase. Thus while Cwp2p was reported to be the most efficient tag
among many tested by Van der Vaart et al. (1997),
the anchoring efficiency is not consistent. A certain portion of the protein
appears to be able to detached from the cell surface, or failed to be transported
to the cell wall and remain in the cytoplasm. This could be due to inefficiency
of the sucrose isomerase signal sequence in targetting the fusion protein to
the cell wall, or failure of the Cwp2p tag to anchor firmly on the cell surface.
The latter could be a result of using only 67 amino acid at the C-terminal of
Cwp2p, or due to interference from the fusion amylase enzyme. By adding a spacer
sequence between the anchor and the tag, (Breinig and Schmitt,
2002) were able to enhance anchoring.
The carboxy terminal of Cwp2p was functional as an anchor protein for the expression of a bacterial enzyme. However, both expression and anchoring efficiency was variable. Further, optimization will be required before the system can be used in industrial processes.
The authors would like to thank the RMI, UiTM for supported work under a short term grant and the NSF, MOSTI for a scholarship to Nadzarah A.Wahab.
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