J. Biol. Macromol., 8 (1), 3-10 (2008)

Review

Novel application of yeast molecular display system to analysis of proteinfunctions
Michiko Kato-Murai* and Mitsuyoshi Ueda
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa
Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
Received October 25, 2007; Accepted October 30, 2007

This review fundamentally covers the functional analysis of proteins and the preparationof modified proteins for the creation of novel functions using a yeast molecular display system
as a molecular tool. Yeast molecular display is a genetic technique of producing proteins or
peptides on the yeast cell surface, such as the cell wall or cell membrane. In this review, recent
studies on the yeast molecular display system in the field of not only applied studies but also
fundamental studies are described with illustrations.
Keywords: molecular display, protein functions, preparation of modified proteins, high-throughput
screening

Introduction
A molecular display system is used forprotein production with which proteins or
peptides can be displayed on the cell surfaceof microorganisms. In this system, proteinsor peptides are displayed as fusion forms
with some anchoring proteins. There aresome display systems using microorganisms,
such as phages (1, 2), bacteria (3-5), andyeasts (6).
In the case of the yeast
Saccharomyces cerevisiae, α-agglutinin (7),a-agglutinin (8), or flocculin (9) has beenused as an anchor protein. These proteins
exist on the yeast cell surface and haveglycosylphosphatidylinositol (GPI) anchorsthat play important roles in the surface
localization of proteins (10).
The
GPI-anchored proteins translocate to the cellsurface through the secretory pathway of S.
cerevisiae (11). The GPI attachment signal
is assigned to the C-terminus of
GPI-anchored protein.
The advantage of using yeast in adisplay system is that it is a eukaryote.
Proteins produced by yeast cells with aqualitycontrolsystemundergopost-translational modification and proteins
having large molecular masses could be
displayed on the cell surface, as comparedwith display systems using phages andbacterial cells.
Furthermore, effective
strategies using the yeast molecular display
system for rapidly analyzing, improving or
modifying the activities and functions ofFig. 1. Construction of plasmid to display thedesired protein and transportation to cell wall of
yeast cell.

*Corresponding author: Dr. Michiko Kato-Murai
Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan
Tel: +81-(0)75-753-6495, Fax:+81-(0)75-753-6112, Email: mk@kais.kyoto-u.ac.jp

4Novel application of yeast molecular display system

Fig. 2. Development of molecular display
system using yeast to fundamental andpractical studies.

Fig. 3. Comparison of conventional systems andyeast molecular display system for protein
production and purification.

proteins can be established, because
protein-displaying cells are used as asingle-protein cluster and multiple protein
purification procedures can be avoided. In
the case of using α-agglutinin as an anchor
protein, active proteins and peptides are
displayed on the yeast cell surface in theform of fusion proteins linked to the
C-terminal half of α-agglutinin, which is aserine- and threonine-rich region andcontains a GPI-anchor attachment signal at
the C-terminus (7). The protein synthesized
and attached to the GPI anchor in theendoplasmic reticulum (ER) is transported tothe plasma membrane by the conventional
vesicular transport system, via the Golgi
apparatus. Part of the GPI anchor of the
GPI-anchored protein transported to theplasmamembraneiscleavedbyphosphatidylinositol-specific phospholipase
C (PI-PLC) and transferred to the outermost
surface of the cell wall. The GPI-anchoredprotein transported to the cell surface is fixedby the addition of β1,6-glucan to the residual
GPI anchor ((12) and Fig. 1). Since 104-105
molecules can be easily displayed on one
yeast cell in many cases (13, 14), it ispossible to consider a protein- or
enzyme-displaying yeast cell as a protein
cluster and a whole-cell biocatalyst in thecase of an enzyme in particular. The yeast
molecular display system has been used todate in various applications (15-17) (Fig. 2).
The principle of this system and some
applications were summarized in detail in aprevious review (18).
In this article, we deal with thefundamental and application studies using theyeast molecular display system.
1. Application to fundamental studies
1-1. Construction of novel protein
purification systemInconventional
overproduction
systems for protein preparation, there aremainly the intracellular production systemand extracellular secretion production system.
However, these conventional methods requirea series of chromatography procedures,
which are time-consuming and tedious.
Furthermore, a refolding procedure isadditionally required when a protein is
produced as an inclusion body. Since it isnecessary to prepare many mutant proteins,to clarify the relationship between structureand functions of them, these conventional
procedures are also labor-intensive and timeconsuming. On the other hand, the yeast
molecular display system is an effective tool
for rapidly analyzing, improving, or
modifying functions of proteins, since protein
displaying cells are used as protein clusters
and many laborious protein-purification
procedures described above can be avoided

Kato-Murai and Ueda

(Fig. 3). If the protein displayed on the cellsurface could be cleaved, it would bepossible to isolate the protein easily without
requiring
conventional
purification
procedures.
To cleave proteins displayed on theyeast cell surface, the selective cleavage
system of GFPuv (19) as a model protein wasconstructed.
A cell-surface-displaying
vectorharboring
genes
of
the
GFPuv-α-agglutinin
fusionproteincontaining the amino acid sequence-Ile-Glu-Gly-Arg-Lys- recognized by theblood coagulation factor, factor Xa, between
GFPuv and α-agglutinin was constructed(Fig. 4a) (20).
The factor Xa is asite-specific serine-type endoprotease that
preferentially cleaves the C-terminal peptide
bond of the recognition sequence. The
display of the fusion protein (GFPuv-factor
Xa recognition sequence-α-agglutinin) on thecell surface was confirmed by the GFPuv
fluorescence around the yeast cells. Whenthe fusion-protein-displaying yeasts weretreated with the factor Xa, the fluorescence of
GFPuv disappeared, indicating that the
GFPuv was successfully cleaved from thecell surface (Fig. 4b). Furthermore, thecleavage of GFPuv from the cell surface wasconfirmed by quantifying GFPuv using achemiluminescencesystemandbyelectrophoresis. This additional system tothe molecular display system makes it easy toisolate and purify the desired protein without

5

many purification procedures.
It issuggested that this system of introducing theprotease recognition sequence between thedesired protein and the anchor protein has theattractive potential for use as a novel protein
purification method instead of theconventional purification method.
1-2. Analysis of processing system of
vacuolar proteases
The vacuole of S. cerevisiae has
several physiological functions, including
proteolysis, pH regulation, osmo regulation,and the storage of amino acids, ions, andpolyphosphates (21, 22). Proteolysis is oneof the very important functions of the vacuole.
In yeast cells, as in mammalian cells (23), thetransport pathway is defined as leading fromthe site of synthesis, the endoplasmic
reticulum (ER), through the Golgi apparatus
to the lysosomal component, the vacuole.
In general, vacuolar hydrolytic enzymes aresynthesized in the form of inactive precursors,which upon arrival at the vacuole areproteolytically cleaved to yield mature active
enzymes, so as not to digest themselves andother proteins before arrival at the destinedorganelle (Fig. 5a). Proteinase A (PrA, EC
3.4.23.25) encoded by PEP4 is alsosynthesized as an inactive preproenzyme andit contains information for its targeting to thevacuole via the secretory route (24, 25).
PrA plays an essential role in the processingof vacuolar hydrolases (26) including

Fig. 4. A image of display of GFPuv on the cell surface and strategy for selective cleavage (a)and demonstration of cleavage of GFPuv from cell surface by protease (b).

6Novel application of yeast molecular display system

Fig. 5. Compartment of the main vacuolar proteases and their processing cascade (a) and strategy
for processing analysis of PrA by altering the transport route of CPY (b).
Active forms areindicated by an asterisk. Solid and dotted lines in the vacuole show the conversion to active enzymes
and activation, respectively. The “ag” of CPYag and proCPYag indicates the fusion proteins with
α-agglutinin.

proteinase B (PrB, EC 3.4.21.48),
carboxypeptidase Y (CPY, EC 3.4.16.5),
alkaline phosphatase, and aminopeptidases.
Although the mechanism of eachprocessing of PrA, PrB, and CPY has beenreported, where their proteases undergo
processing to be converted into mature
enzymes remains unclear. In particular, thelocation of maturation of proproteinase A(proPrA), which is a precursor of PrA still
remains to be solved. PrA is a key enzyme
as an initiator of a processing cascade forvacuolar proteases.
Although it waspredicted that the conversion of inactive
proPrA to active PrA including pseudoPrA
occurs autocatalytically owing to the acidic
pH in the vacuole (26-28), it was reported
that vacuolar acidification was not necessary
for zymogen activation by the experimentusing vacuolar H-ATPase mutant yeast strain
in which the vacuolar pH is neutral (29).
To demonstrate the location of proPrA
activation, a processing analytical systemusing yeast molecular display wasconstructed.
The plasmid containing
procarboxypeptidase Y (proCPY)-encoding
gene for displaying the proCPY on the yeast
cell surface as the proCPY-α-agglutinin
fusion protein (proCPYag) was introduced invarious protease-knockout yeast strain.
ProCPYag produced in the cell changes thetransport pathway to the cell surface, not tothe vacuole.
The phenotype by the
introduced gene was analyzed by measuring
the activity of CPY displayed on the cellsurface (Fig. 5b). The activity of CPY
displayed on the cell surface was measured
after the conversion of inactive proCPYag toactive CPYag by treatment of whole cellswith proteinase K, which converts proCPY to
CPY in vitro (30) (Fig. 6a). When proCPY
wasexpressedinthewild-type,
CPY-knockout, and PrB-knockout strains,
CPY was displayed in its active form (mature
CPY), but in the PrA-knockout strain, CPY
was present in its inactive form (proCPY).
That is, CPY was displayed in its active form
only when PrA existed in cells (Fig. 6b).
PrA was not transported to the extracellular
medium, as determined by measuring theactivity of PrA in the extracellular fraction.
Furthermore, PrA is an aspartic protease witha structural similarity to other aspartic
proteases such as cathepsin D (31), alysosomal enzyme in higher eukaryotes.
Cathepsin D is present in its active form in
the endosome (32), which is the transportcompartment from the Golgi apparatus to thelysosome.
These findings indicate thatinactive proPrA had already been converted
into active pseudoPrA or PrA before its
transport to the vacuole and that active PrA
might convert proCPY into CPY before thetransport of proCPY to the vacuole. The

Kato-Murai and Ueda

7

Fig. 6. Measurement procedure for activities
of proCPY or CPY displayed on the various
proteases-deficient strains with or without
proteinase K (PrK) treatments (a) andputative processing by PrA (b). The “ag” of
CPYag and proCPYag indicates the fusion
protein with α-agglutinin.

results obtained using the yeast moleculardisplay system, suggest that the autocatalytic
activation to active PrA might occur during
the early transport to the vacuole from theGolgi apparatus (Fig. 6b).

2. Application to practical studies
2-1. Preparation of whole-cell biocatalysts
Lipases are used in many applications
in chemical processing of organic solventsand detergent formulations, the synthesis of
biosurfactants, the dairy industry, paper
manufacture, nutrition, cosmetics, andpharmaceutical processing (33). Lipases
can be effective for asymmetric synthesis byutilizing their high stereo-specificity inchemical synthesis and for esterification in
preparing esters from fatty acids by taking
advantage of their substrate specificity (inparticular,
chain-length
specificity).
Candida antarctica lipase B (CALB) (34) is
one of the most widely used biocatalysts (35)and the present industrial preparations of
CALB are Novozym 435 (Novozymes A/S,
Bagsvaerd, Denmark) and Chirazyme L-2(Roche Molecular Biochemicals, Mannheim,
Germany) as immobilized products, but they
are expensive (36).
To easily prepare
CALB at a low cost, CALB was displayed on
the cell surface using a yeast moleculardisplay system (13). When CALB wasdisplayed, it showed a preference foradvantageous for producing flavors, although
Rhizopus oryzae lipase (ROL) preferred fatty
acids with intermediate lengths (37).
Furthermore, CALB displayed on the cellsurface showed high thermal stability.
These
findings
suggestthata
CALB-displaying yeast strain is also
practical for industrial use as a whole-cellbiocatalyst.
When ROL-displaying yeasts wereused in synthesis reactions, they could
catalyze synthesis reactions in organic
solvents. The activity of ROL-displaying
yeasts used for esterification with palmitic
acid and n-pentanol in heptane containing
0.2 % H2O was 3.8×104-fold higher than thatof a commercial lipase (38). To increase
lipase activity, mutation was introduced into
ROL by the error-prone PCR method, which
has been widely used to improve enzyme
functions, in combination with the moleculardisplay system. Some of the ROL mutants
showed activities 3- to 6-fold higher than theoriginal ROL-displaying yeast.
The
combination of mutation introduction and theuse of the molecular display system opens a
new door to obtaining proteins or enzymes
with higher activities.

8

Novel application of yeast molecular display system

2-2. High-throughput screening system ofmodified enzymesIn
recentyears,
asmaterial
technologies
includingnanoandmicro-technologies
haveprogressively
advanced, some high-throughput screening
systems have been developed.
In theconventional screening method in which agar
plates are used to select colonies on the basisof halo formation, colonies forming a halo
are recultivated and then their functions andbiochemical properties are analyzed in detail.
Such a method requires large amounts of
medium and considerable effort and time,
particularly in the screening of numerous
mutants.
Furthermore,thefluorescence-activated cell sorting (FACS)
system, which is used for the screening ofclones with high activities, permits the rapid
quantitative and selective isolation andevaluation of significant clones. By using
FACS, target cells are detected only byfluorescence of labeled chemicals bound totarget cells, and then can be separated. To
screen easily and to obtain a desired single
cell, a novel screening system for a single
cell was constructed using a yeast cell
microchamber chip in combination with themolecular display system (39).
It was possible to place a single yeast
cell into each microchamber, to observe thebehavior of a single cell, to select the target
cell, and to cultivate a single cell in eachmicrochamber, which was made possible by
the prevention of the rapid drying of themedium (40).
Using a ROL-displaying
yeast, the hydrolytic reaction of a single cell
could be detected in a microchamber.
Furthermore, the ROL-encoding gene wasamplified by PCR and then its sequence wasconfirmed using a DNA sequencer. These
results demonstrate that this yeast cell chip in
combination with the molecular display
system may be used as a tool in ahigh-throughput screening system not only
for a single living cell and a whole-cellcatalyst but also for functional protein
molecules from protein libraries displayed on
the cell surface (39).
Further directions
A molecular display system using yeast
S. cerevisiae strain enables the display oflarge protein molecules on the cell surface, incomparison with systems using bacterial cells
and phages. Yeasts can produce foreign

Fig. 7. Comprehensive analysis and screening of displaying-proteins using yeast cell
microchamber chip.

Kato-Murai and Ueda

proteins with correct conformations in theform of undergoing the post-translational
modification. The display of peptides orproteins on the yeast cell surface endows
yeasts with novel functions.
With the development of genome
analysis,numerous
single-nucleotide
polymorphisms (SNPs) have been found in agenome sequence. These SNPs seem to berelated to individual differences, such as
responsiveness to medicine and susceptibility
to a disease. By examining the structureand function of mutant or modified proteins
generated from protein-encoding genes
containing SNPs, the use of a “tailor-mademedicine” suitable for a specific person or
patient will become possible.
Thedevelopment of methods of analyzing SNPs
rapidly will be important to realize this goal.
To analyze protein structure-function
relationships, it is required to prepare various
mutant proteins. By using the moleculardisplay system, the preparation of many
protein libraries becomes easy and a rapidfunctional analysis of proteins becomes
possible because protein-displaying yeasts
are used as single protein clusters without
requiring conventional protein purification
procedures.
Furthermore,thehigh-throughput system coupled with theyeast molecular display system permits thescreening for target cells with higher
activities or novel functions (Fig. 7). Thisyeast molecular display system will be
widely applicable in scientific fields as amolecular tool to prepare various proteins,
easily, and rapidly.

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Communicated by Ueno Hiroshi