Microbial cell-surface display

Sang Yup Lee
1,2
, Jong Hyun Choi
1,2
and Zhaohui Xu
1
1
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering
and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong,
Yuseong-gu, Daejeon 305-701, South Korea
2
Center for Ultramicrochemical Process Systems, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong,
Yuseong-gu, Daejeon 305-701, South Korea
Cell-surface display allows peptides and proteins to be
displayed on the surface of microbial cells by fusing
them with the anchoring motifs. The protein to be dis-
played – the passenger protein – can be fused to an
anchoring motif – the carrier protein – by N-terminal
fusion, C-terminal fusion or sandwich fusion. The
characteristics of carrier protein, passenger protein and
host cell, and fusion method all affect the efficiency of
surface display of proteins. Microbial cell-surface display
has many potential applications, including live vaccine
development, peptide library screening, bioconversion
using whole cell biocatalyst and bioadsorption.
The first surface expression system was developed by
George P. Smith in the mid-1980s, who displayed – on the
surface of bacteriophage – the peptides and small proteins
fused with the pIII protein of the filamentous phage [1].
Since then, various phage display systems have been
developed to express foreign proteins on the surface of the
phage. However, the size of foreign protein to be displayed
on the surface of phage is rather limited [2]. The microbial
cell-surface display system was developed to solve this
problem and for several other unique applications. Micro-
bial cell-surface display is carried out by expressing a
heterologous peptide or protein of interest (the passenger
or target protein) as a fusion protein with various anchor-
ing motifs, which are usually cell-surface proteins or their
fragments (carrier proteins). Depending on the character-
istics of passenger and carrier proteins, C-terminal fusion,
N-terminal fusion or sandwich fusion strategy can be
considered.
Microbial cell-surface display has a wide range of bio-
technological and industrial applications (Fig. 1) includ-
ing: live vaccine development to expose heterologous
epitopes on human commensal or attenuated pathogenic
bacterial cells to elicit antigen-specific antibody responses
[3,4]; screening-displayed peptide libraries by sequential
binding and elution or, more efficiently, by fluorescence-
activated cell sorting [5,6]; antibody production by express-
ing surface antigens to raise polyclonal antibodies in
animals [7]; bioadsorbents for the removal of harmful
chemicals and heavy metals [8–11]; whole-cell biocatalysts
by immobilizing enzymes [12]; biosensor development by
anchoring enzymes, receptors or other signal-sensitive
components for diagnostic, industrial or environmental
purposes [13,14]; anddetectionof single amino acidchanges
in the target peptides after random mutagenesis [15].
There are several reviews on the applications of cell sur-
face display [16–19] – here, we review recent advances in
the development of microbial cell-surface display systems,
focusing on the characteristics of carrier and passenger
proteins affecting the efficiency of surface display.
Carrier protein (anchoring motif)
During the 1990s, many different carrier proteins (anchor-
ing motifs) were developed for the surface display of pro-
teins. The use of different carriers often results in different
physiological effects on host cells. For example, using
proteins that are essential for cellular functions or struc-
tures, such as outer membrane proteins and subunits of
cellular appendages, might lead to growth defects and
destabilization of cell envelope integrity. A successful
carrier should meet the following four requirements: it
should have an efficient signal peptide or transporting
signal to allow premature fusion protein to go through the
inner membrane; it should have a strong anchoring struc-
ture to keep fusion proteins on the cell surface without
detachment; it should be compatible with the foreign
sequences to be inserted or fused (i.e. the carrier should
not become unstable on the insertion or fusion of hetero-
logous sequences); and it should be resistant to attack by
proteases present in the periplasmic space or medium.
Each type of carrier has different characteristics and
might thus be useful for specific applications. For example,
bacterial fimbriae, S-layer proteins, ice nucleation pro-
tein (INP) and some outer membrane proteins (such as
Escherichia coli TraT) are efficient carriers for immuno-
stimulation purposes [3,20,21] and are therefore particu-
larly useful for developing recombinant vaccines.
The location in the carrier for the insertion or fusion of
peptide or protein to be displayed is important because it
influences the immobilization efficiency, stability, specific
activity and post-translational modification of the fusion
protein. For example, four positions in FimA (amino acids
at positions 25, 45, 80 and 105) were examined as fusion
sites for inserting the neutralizing epitopes of the cholera
toxin Bchain. Three positions (amino acids at positions 25,
45 and 80) were compatible but one of them (amino acids
at position 105) was not [22]. In another example, 11
potentially permissive sites of Caulobacter crescentus
S-layer protein were examined as fusion sites to display
heterologous peptides but nine of them resulted in Corresponding author: Sang Yup Lee (leesy@mail.kaist.ac.kr).
Review TRENDS in Biotechnology Vol.21 No.1 January 2003
45
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proteolysis of the fusion peptides [23]. Therefore, it is
important to identify the best fusion or insertion site
between the carrier and the passenger proteins.
Because the protein to be displayed should face towards
the surrounding medium, it is necessary to identify the
regions of the carrier protein that are exposed outside the
cells. There are several ways to do this, the simplest, and
most efficient, method is homology comparison. Aligning
the sequence of the carrier protein with its homologous
variants of known structure can suggest potential expo-
sure sites. Xu and Lee used this approach to identify eight
external loops of E. coli OmpCand successfully used one of
them to display poly-histidine peptides [11]. Another
method is calculating the hydropathy profile of the carrier
using predictive algorithms or computer programs [24]. If
these methods cannot provide answers, another, experi-
mentally more demanding, method can be used: reporter
sequences can be inserted randomly into the primary
sequence of the carrier protein to detect the presence of
reporter protein on the cell surface.
Passenger protein (target protein)
The passenger protein to be displayed is selected by the
required application. However, it should be noted that
passenger proteins themselves also influence the trans-
location process and final surface display. Different pas-
senger proteins fused to the same carrier protein are
transported to the different locations in the same host
strain [25]. The characteristics of passenger proteins are
known to affect transportation process significantly. The
folding structure of the passenger protein (such as the
formation of disulfide bridges) at the periplasmic side of
the outer membrane can affect its translocation [26,27]. In
addition, the insertion of amino acid sequences containing
many charged residues or hydrophobic residues results in
inefficient secretion in bacteria. Passenger protein con-
taining four phenylalanine residues could not be displayed
on the cell surface because of inefficient secretion [28].
However, the protein could be displayed successfully when
phenylalanine residues were either deleted or substituted
by serine residues [28]. However, when this strategy of
changing amino acid sequences is used, it should be noted
that the alteration of the biological function of the
passenger protein might become a new problem.
Host strain
Selection of a host strain for surface display is an
important factor that cannot be neglected. A good host
should be compatible with the protein to be displayed and
should be easy to cultivate without cell lysis. Also, the host
strain should have lowactivities of cell wall associated and
extracellular proteases. It was shown that cholera toxin B
subunit (CtxB) was displayed successfully in an ompT
(encoding the outer membrane protease T, OmpT) nega-
tive strain but was released into the mediumin a wild-type
strain possessing intact OmpT [26,27]. For Gram-negative
bacteria, including E. coli, the fragility of outer membrane
caused by the display of proteins can be a problem.
Nevertheless, E. coli is still an attractive host because the
availability various genetic tools and mutant strains and
the high transformation efficiency makes it an ideal host
for screening a large peptide or protein library after
surface display.
Gram-positive bacteria seem to be more suitable for
whole-cell catalysts and whole-cell adsorbents because of
the rigid structure of their cell walls; Bacillus and
Staphylococcus strains have been used most often. Screen-
ing of peptide and protein libraries displayed on the sur-
face of Gram-positive bacteria has not yet been reported.
Saccharomyces cerevisiae is a good host for surface display
of proteins and has several advantages over other bacteria.
First, it is generally recognized as safe, which allows its
use in food and pharmaceutical applications. Second, its
protein folding and secretory machineries are similar to
those of mammalian cells, which allows display of mam-
malian proteins better than bacterial system. Third, its
fermentation characteristics are well-known and fourth,
passenger proteins can be displayed by linking to the cell
wall via a glycosyl phosphatidylinositol (GPI) anchor or by
disulfide bonds.
Surface display systems developed for Gram-negative
bacteria
Gram-negative bacteria possess a complex cell envelope
structure that consists of cytoplasmic membrane, peri-
plasm and outer membrane. This means that the surface-
anchoring motif, fused with the protein to be displayed,
should pass through the cytoplasmic membrane and
periplasm to the outer membrane. The targeting and
anchoring mechanisms of carrier proteins vary among
the different surface proteins and different approaches
have been used to develop successful display systems.
As shown in Table 1, most of carrier proteins developed
to date are based on the outer membrane proteins.
Hoischen et al. [29] described a novel system that allows
display of recombinant proteins on the cytoplasmic
membrane using the L-form cells of E. coli and Proteus
mirabilis. Fig. 2a shows various anchoring motifs that
have been used for the surface display of proteins in Gram-
negative bacteria.
Various gene fusion strategies can be considered in
Gram-negative bacteria. The N-terminal fusion approach
is suitable when the carrier protein possesses a directing
Fig. 1. Applications of microbial cell surface display.
TRENDS in Biotechnology
Oral vaccines
Screening of peptide libraries
Whole-cell
biocatalyst for
bioconversion
Bioadsorbent
Biosensor
Mutation detection
Antibody production
Cytosol
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and anchoring domain in its C-terminus part. Peptidogly-
can-associated lipoprotein (PAL) is a typical carrier pro-
tein of this type. PAL binds to the peptidoglycan layer with
its C-terminal portion and to the outer membrane with its
N-terminal cysteine modified by a lipid moiety [13].
Members of the immunoglobulin A (IgA) protease family
contain C-terminal autotransporter structures that pro-
mote the translocation of the N-terminally attached
passenger domains across the outer membrane [27]. The
C-terminal domain forms porin-like b-barrel channels on
the outer membrane to facilitate the transportation of the
N-terminal passenger domain. E. coli adhesion protein
AIDA-I was used as an anchoring motif to display dimeric
bovine adrenodoxin (Adx) on the cell surface. This system
can be used for whole-cell steroid bioconversion [30]. When
the cholera toxin B subunit (CtxB) was displayed using
this system, the passenger domain was released from the
cell surface by protease cleavage within the linker space
[26]. Shigella VirG protein, which is responsible for the
deposition of filamentous actin, has been used as an
anchoring motif for displaying PhoA and MalE on the
surface of E. coli [24].
Several outer membrane proteins carry targeting
sequences on their N-termini. These proteins can be
used as carrier proteins to construct display systems by
C-terminal fusion method. The Lpp–OmpA hybrid is a
Table 1. Representative display systems for the expression in Gram-negative bacteria
Carriers (size) Passengers (size) Hosts Applications Refs
N-terminal fusion
Neisseria gonorrhoeae IgA1
protease (45 kDa)
CtxB (13 kDa) E. coli (ompT
2
) Vaccines [27]
E. coli adhesin (AIDA-I)
(51.5 kDa)
CtxB (13 kDa) E. coli (ompT
2
) Vaccines [26]
Dimeric Adx (14.4 kDa) E. coli Whole-cell steroid synthesis [30]
Shigella VirG (37 kDa) E. coli MalE, PhoA (471 aa) E. coli (ompT
2
) VirG export pathway study [24]
E. coli PAL (173 aa) Anti-atrazine antibody fragment (252 aa) E. coli Biosensor [13]
Domain B of SpA (77 aa) GFP-K ras and GFP-p53
a
E. coli Detection of single amino acid
changes
[15]
C-terminal fusion
E. coli Lpp-OmpA (123 aa) Organophosphorus hydrolase (365 aa) E. coli Biodegradation [12]
Pytochelains (40 aa) E. coli Adsorption (bioaccumulation) [8]
PE DIII antgen, extracellular domain
of human ErbB2 and IL2-Ra (237 aa)
E. coli Selection of phage antibody [44]
GFP (239 aa) E. coli Improve efficiency reporter gene [45]
Pseudomonas syringae INP
(36 kDa)
CMCase (33 kDa) E. coli Whole-cell biocatalysts [32]
Levansucrase (424 aa) E. coli Utilization of levan [31]
CMCase mutant library (33 kDa) E. coli Development of protein, screening [46]
HbsAg (168 aa) S. typhi Ty21a Oral vaccines [3]
Salmobin (26 kDa) E. coli Display of eukaryotic protein [33]
Synthetic phytochelatin (40 aa) E. coli Bioadsorption [9]
OPH (365 aa) E. coli Screening of OPH variants [34]
OPH (365 aa) Moraxella sp. Biocatalysts [47]
E. coli TraT (26.5 kDa) HbsAg
a
E. coli Vaccines [20]
RHO (7.5 kDa) E. coli Bacteria-host interaction study [21]
E. coli intimin EaeA (659 aa) EETI-II, interleukin 4, Bence-Jones protein
REI (128 aa)
E. coli Protein translocation study [48]
Sandwich fusion
E. coli OmpC (367 aa) Poly-His peptides (162 aa) E. coli Heavy metal removal [11,49]
Vibrio cholerae OmpS (390 aa) Epitopes from staphylococcal FnBPA,
E. coli PapG (115 aa)
E. coli Vaccines [50]
E. coli LamB (446 aa) HMT, YMT (66 aa) E. coli Heavy metal removal [10]
HbsAg
a
E. coli Vaccines, antibody production [7]
Evolution variants for selection E. coli Screening of variants (CABS) [51]
E. coli TraT (26.5 kDa) Epitope from polio virus
a
E. coli Peptide [36]
E. coli FliC (498 aa) Adhesive domains of staphylococcal
FnBPA and Yersinia YadA (115 aa)
E. coli Expression of adhesive peptides [6]
E. coli FimH (30 kDa) Random peptide library (33 aa) E. coli Screening of binding motifs [52]
E. coli FimA (180 aa) CtxB epitopes (34 aa) E. coli Vaccines [22]
E. coli F pilin (7.5 kDa) Peptide (15 aa) E. coli Selective phage infection [53]
Caulobacter crescentus RsaA
(1073 aa)
Fragment from Pseudomonas
aeruginosa K pilin (12 aa)
C. crescentus Not indicated [23]
ETC
LacY(15.4 kDa), SecY (8.3 kDa) Staphylokinase (SAK) (136 aa) E. coli Specific adhesin-receptor interaction [29]
CcmA (5.1 kDa) Staphylokinase (SAK) (136 aa) Proteus mirabilis Studies
Abbreviations: Adx, Bovine adrenodoxin; CABS, continuous affinity-based selection; CtxB, cholera toxin B subunit; EETI-II, Ecballium elaterum trypsin inhibitor II; PAL,
peptidoglycan associated lipoprotein; CMCase, carboxymethylcellulase; HBsAg, hepatitis B virus surface antigen; FnBPA, fibronectin binding protein A; GFP, green
fluorescent protein; poly-His, poly-histidines; HMT, human metallothionein; PE DIII, catalytic domain III of Pseudomonas aeroginosa exotoxin; RHO, snake venom
rhodostomin; YMT, yeast metallothionein; TGEV, transmissible gastroenteritis virus; SpA, Staphylococcal protein A; sc, single chain; Fv, variable fragment.
a
Size information not available.
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good example of this type. An Lpp–OmpA chimera
consists of the signal sequence and first nine N-terminal
residues of the mature E. coli lipoprotein, and the residues
46–159 or 46–66 of the E. coli outer membrane protein A
(OmpA) [8,12]. The Lpp part is necessary for proper
localization to the outer membrane; the OmpA part is
responsible for the transportation of foreign proteins fused
at the C-terminus across the outer membrane.
Pesudomonas syringae ice nucleation protein (INP) is
another popular anchoring motif that has been used
successfully to display by C-terminal fusion several pro-
teins, including levansucrase [31], carboxymethylcellulase
(CMCase) [32], salmobin[33] andorganophosphorushydrol-
ase (OPH) [34]. INPis an outer membrane protein found in
Erwinia, Pseudomonas and Xanthomonas. It nucleates ice
formation in supercooled water and causes frost injury to
plants. Its internal repeats serve as templates for ice
nucleation and the length of this region is adjustable in-
frame [31,32]. It will therefore be possible to display pep-
tides or proteins using INP motifs of different lengths,
which increases the likelihood of avoiding potential steric
hindrance among the displayed proteins. Whereas most
cell-surface display systems are limited in the size of
foreign protein that can be expressed, the INP-based
system can express proteins as large as 60 kDa (Fig. 2b).
Klebsiella pneumoniae pullulanase, an extracellular
enzyme, stays on the cell surface temporarily by means of
the fatty acid attached to its N-terminal cysteine and is
gradually released into the medium[35]. b-Lactamase and
alkaline phosphatase are displayed by the C-terminal
fusion method using pullulanase as an anchoring motif.
However, pullulanase seems to be unsuitable as a carrier
protein in most cases, unless its release from the cell
surface can be prevented.
Sandwich fusion is the most commonly used strategy for
the surface display of proteins in Gram-negative bacteria.
Three classes of proteins have been used as carrier pro-
teins: outer membrane proteins (OMPs), subunit proteins
of extracellular appendages and S-layer proteins.
OMPs form transmembrane b-barrels on the outer
membrane. The b-Barrels are composed of antiparallel
b-strand pairs connected by short loops on the periplasmic
side and by long loops on the external side. The external
loops are generally less conservative and therefore seemto
be tolerant to a certain degree of modification, such as
substitution, insertion and deletion. These external loops
can potentially be used as fusion sites for the display of
heterologous proteins. It has generally been believed that
the external loops of OMPs could only accept foreign
peptides of 70 amino acids or less owing to the disruption of
membrane integrity of carrier protein [18]. This limitation
also applies when bacterial surface appendages are used
as sandwich fusion partners. However, it has more
recently been demonstrated that the E. coli OmpC could
be used as a sandwich fusion partner, displaying much
longer polypeptides of 162 amino acids, which is the
largest peptide inserted to date using the sandwich fusion
method [11] (Fig. 2c). The E. coli LamB, which is a
TRENDS in Biotechnology
Outer
membrane
Plasma
membrane
S-layer
protein
Flagellar
Pilin
Lipoprotein
Outer
membrane
protein
IgA protease
Passenger
protein
(a)
Lpp-OmpA fusion
PUL complex
Passenger
protein
Pullulanase
Ice nucleation
protein (INP)
LacY, SecY
Cytosol
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L1 L2 L3 L4 L5 L6 L8
L7
LQVDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLD
PSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLEDILQSKG
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E. coli OmpC
(c)
(b)
Outer
membrane
Internal
repeated
domain
Passenger
Passenger
N-terminal
anchoring
domain

Poly-histidine linker
Fig. 2. Cell surface display systems in Gram-negative bacteria; green circles represent heterologous passenger proteins. (a) Surface display systems developed in Gram-
negative bacteria. The patents describing these display systems are as follows: S-layer protein (US5874267), OmpC (US6274345), PhoA (US535697), OprF (WO9324636),
OmpA (DE4243770, EP0474891), lipoprotein (WO950479), IgA protease (WO9735022), Pilin (WO9410330), Lpp–OmpA (WO9310214, WO9849286), INP (WO9737025,
WO9967366, WO0246388) and Flagella (WO006010). (b) Cell-surface display system using ice nucleation protein (INP), which is a representative example of the N-terminal
fusion method. The INP is the most stable and useful carrier to express foreign proteins as large as 60 kDa. (c) Cell-surface display system using E. coli outer membrane pro-
tein C, which is a representative example of sandwich fusion method. In this system, poly-histidine (poly-His) peptides of up to 162 amino acids could be inserted into the
seventh external loop (L7) of OmpC and could be efficiently exposed on the E. coli cell surface.
Review TRENDS in Biotechnology Vol.21 No.1 January 2003
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transport protein for maltose and maltodextrin, was also
used as a sandwich-anchoring motif. However, only short
polypeptides of up to 88 amino acids could be displayed
[7,10]. Some carrier proteins can display foreign peptides
at more than one fusion site – E. coli TraTis such a carrier
protein. Passenger peptides could be fused not only to the
middle of TraT [36] but also to the C-terminus [20,21].
When the snake venom rhodostomin, a distintegrin, was
displayed on the surface of E. coli using TraT, recombinant
E. coli adhered to and internalized into BHK-21 hamster
cells [21]. OmpC can also be used as an anchoring motif,
accepting the passenger proteins both by sandwich fusion
[11] or by C-terminal fusion (Lee et al. unpublished
observations).
The subunit proteins of E. coli flagella and fimbriae (or
pili) can be used to display heterologous proteins. Some of
the exposed sites of the major subunit proteins are dis-
pensable and are relatively tolerant in accepting hetero-
logous sequences. The chimeric subunits carrying foreign
polypeptides can still be assembled into polymeric append-
ages providing each of them carrys a foreign peptide
segment [6,22]. Bacterial flagella and fimbriae are strongly
immunogenic as a result of their polymeric and pertina-
cious nature, which is useful for carrying various epitopes
(Table 1).
The S-layer protein of the Gram-negative bacterium C.
crescentus was found to contain an N-terminal domain,
which can bind to the outer membrane, and a C-terminus
carrying a secretion signal [37]. This S-layer protein was
used to display a peptide of 12 amino acids from Pseudo-
monas aeruginosa K pilin in C. crescentus [23].
Surface display systems developed for Gram-positive
bacteria
Many surface proteins of Gram-positive bacteria are
covalently immobilized to the cell wall, typically involving
a specific C-terminal sorting signal consisting of 32–38
amino acids. Staphylococcal protein A(SpA) has oftenbeen
used as a model system to study anchoring mechanisms of
surface proteins in Gram-positive bacteria (Fig. 3a). The
sorting signal includes an LPXTG (in single-letter amino
acid code, where Xdenotes any amino acid) sequence motif
followed by a stretch of ,23 hydrophobic residues and a
tail of six or seven mostly positively charged residues at
the extreme C-terminus [38]. The LPXTG motif contains a
cleavage site for sortase (SrtA), which is an enzyme of 206
amino acids that cleaves polypeptides between the thre-
onine and the glycine of the LPXTG motif. The hydro-
phobic domain is a membrane-spanning region. The
charged tail serves as a retention signal to prevent secre-
tion of the polypeptide chain into the surrounding medium
[38]. The anchoring domain, which includes a standard cell
wall sorting signal as well as an appropriate cell wall-
spanning region, has been used to display various poly-
peptides and proteins (Fig. 3b, Table 2).
Besides the LPXTG box, some other anchoring motifs
that can be used for displaying proteins on the outer
surface of Gram-positive bacteria have been developed
(Fig. 3b, Table 2). One of these is the S-layer homology
(SLH) domain, which has been found in many Gram-
positive bacteria, especially in the members of Deinococcus-
Thermus phylogenetic group. The SLH domains are
present in singles or in multiples at the N-terminus of
Gram-positive S-layer proteins and consist of residues
of 70 amino acids. They are predicted to be composed of two
a-helices flanking a b-strand. Experimental data suggest
that the SLH domains mediate association of SLH-
domain-bearing proteins to the polymers of the secondary
cell wall, which are linked covalently to the peptidoglycan
layer. The SLH domain of the Bacillus anthracis S-layer
protein EA1 has been used to display tetanus toxin
fragment C in Bacillus anthracis [39].
Surface display systems developed for yeast
The display of foreign proteins on the surface of yeast
provides several unique advantages. Two types of manno-
proteins are present in the cell wall of Saccharomyces
cerevisiae: sodium dodecyl sulfate (SDS)-extractable and
glucanase-extractablemannoproteins. TheSDS-extractable
mannoproteins appear to be associated noncovalently with
the cell wall and can be extracted from the cell wall by
treating with SDS and a reducing agent, such as DTT or
b-mercaptoethanol. The glucanase-extractable mannopro-
teins are thought to be covalently cross-linked to the cell
wall b-glucan and released only after digestion of the cell
wall withb-glucanase. Many glucanase-extractable manno-
proteins have been found to be rich in serine and/or
threonine, and generally contain a putative glycosyl phos-
phatidylinositol (GPI) attachment signal at the C-termini
[40]. The covalent association of these proteins with the
cell wall requires the addition of a GPI anchor to their C-
termini, because this traverses the secretory pathway.
Periplasmic intermediates are produced after the removal
of fatty acid and inositol from the GPI anchor, followed by
the cross-linkage of the intermediates to the b-1,6-glucan.
Thus, when a foreign peptide or protein to be displayed is
fused to a mannoprotein, it will most likely be carried to
and anchored covalently on the cell surface. Table 3
summarizes yeast cell-surface display systems and their
Fig. 3. Cell-surface display systems in Gram-positive bacteria; green circles rep-
resent heterologous passenger proteins. (a) Cell-surface display system using sta-
phylococcal protein A, which is a representative example of the N-terminal fusion
method. (b) Schematic illustration of surface display systems constructed in
Gram-positive bacteria. Several patents are available for using protein A as an
anchoring motif (WO9709437, US5616686, WO9318163, US5958736, WO9640943
and US5821088).
TRENDS in Biotechnology
Protein A,
M6 protein
X M ABP
S-layer protein
Peptidoglycan
ETC
Cell wall
Cytosol
X
M
A
B
P
(a)
Cortex
Spore coat
protein
(b)
Spore
core
Spore coat
Albumin
binding
protein
Charged
repetitive
region
Cell wall
Passenger
Surface-bound
receptor containing
LPXTG motif
Peptidoglycan
Review TRENDS in Biotechnology Vol.21 No.1 January 2003
49
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current applications. Almost all of the cell-surface display
systems developed for yeast are GPI anchor-dependent. S.
cerevisiae a-agglutinin has widely been used to display
various peptides and proteins such as hepatitis B virus
surface antigen, lipase, glucoamylase, green fluorescent
protein (GFP) and blue fluorescent protein (BFP) [14,41].
Some newly identified yeast cell-wall proteins, such as
Cwp1p, Cwp2p, Tip1p, Tir1p/Srp1p and Sed1p, have been
proven capable of displaying of a-galactosidase [40] and
GFP on the surface of S. cerevisiae. Notably, sixfold to
eightfold higher levels of a-galactosidase could be dis-
played by using Cwp2p and Sed1p instead of a-agglutinin
as a carrier protein. More examples of displaying mam-
malian proteins on the surface of yeast are expected to
appear.
A peek at the future development
Despite the successful development of various cell-surface
display systems, problems remain to be solved and
improvements to be made. For example, questions have
been raised over the quality of the peptide library dis-
played on the cell surface because of the potential bias
introduced by the sequence-dependent variation in expres-
sion level. In the development of whole-cell biocatalysts by
Table 3. Representative display systems for the expression in yeast
Carriers (size) Passengers (size) Hosts Applications Refs
N-terminal fusion
Saccharomyces. cerevisiae
a-agglutinin (Aga1p)
a
HBsAg (139 aa) S. cerevisiae Oral vaccines [41]
ZZ domain of
Staphylococcus aureus (130 aa)
S. cerevisiae Whole-cell immunoadsorbents [60]
GFP, BFP (238 aa) S. cerevisiae Detection of glucose concentration [14]
S. cerevisiae Flo1p (344 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae Cwp1p (213 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae Cwp2p (67 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae Tip1p (212 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae Sed1p (229 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae YCR89w (313 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
S. cerevisiae Tir1p (212 aa) a-galactosidase (450 aa) S. cerevisiae Anchoring domain selection [40]
C-terminal fusion
b
S. cerevisiae Aga2p (794 aa) scFv (271 aa) S. cerevisiae Screening of polypeptide libraries [5]
Polypeptide libraries (230 aa) S. cerevisiae Screening for novel binding or
improved mutants
[61]
Abbreviations: BFP, blue fluorescent protein; HBsAg, hepatitis B virus surface antigen; GFP, green fluorescent protein; sc, single-chain; Fv, fragment variable; TCR, T cell
receptor.
a
Size information not available.
b
a-Agglutininis composedof two subunits: Aga1pand Aga2p. Aga1p anchors ontothe cell wall via GPI modificationprocess. Aga2p is linked to Aga1pby twodisulfide bonds,
with its native binding activity on the C-terminus [5].
Table 2. Representative display systems for the expression in Gram-positive bacteria
Carriers (size) Passengers (size) Hosts Applications Refs
N-terminal fusion
Staphylococcus aureus
FnBPB (128 aa)
Lipase, b-lactamase (66 kDa) Staphylococcu
carnosus
In vivo immobilization
of enzymatically active
polypeptides
[42]
Streptococcus pyogenes
M6 (262 aa)
Hornet venom allergen (204 aa) Streptococcus
gordonii
Vaccines [54]
Staphylococcal protein A
(SpA) (472 aa)
Variants of human RSV
glycoprotein fragment
a
S. xylosus Live vaccines [28]
CtxB (103 aa) S. xylosus,
S. carnosus
Live bacterial vaccines [4]
Streptavidin monomer (158 aa) Lactococcus lactis Cell immobilization on
polystyrene support
[55]
Epitopes from fibronectin binding
proteins of Streptococcus
dysgalactiae and S. aureus
(43 aa)
S. carnosus Vaccine delivery systems [56]
Ni binding cellulose binding
domain variants (36 aa)
S. carnosus Screening, whole cell
assay
[57]
C-terminal fusion
Bacillus anthracis S-layer
protein EA1 (209 aa)
Tetanus toxin fragment C(ToxC)
(50 kDa)
B. anthracis Protect against tetanus
toxin
[39]
Bacillus subtilis CotB (380 aa) C-terminal fragment of tetanus toxin
(TTFC) (459 aa)
B. subtilis spore Expression of bioactive
molecules on spore
[58]
Bacillus sphaericus S-layer
protein SbpA (1068 aa)
Short affinity peptide Strep-Tag I (9 aa)
Major birch pollen allergen Bet v1 (17 kDa)
B. sphaericus CCM 2177 Potential application for
biochip developments
[59]
Abbreviations: FnBPB, fibronectin binding protein B; RSV, respiratory syncytial virus; SpA, Staphylococcal protein A.
a
Size information not available.
Review TRENDS in Biotechnology Vol.21 No.1 January 2003
50
http://tibtec.trends.com
cell-surface display, the reduction in the activity of enzyme
is an issue. Compared with their free forms, surface-
anchored a-galactosidase, lipase, cutinase and b-lactamase
show reduced catalytic activities [40,42,43]. The steric
hindrance, incomplete exposure, unfolded or misfolded
structure and repulsion of substrate by the hydrophobicity
of the cell wall were thought to cause this problem [40].
Another important problem in cell-surface display is the
occurrence of artifacts caused by cell envelope changes.
Dhillon et al. [13] successfully targeted functional anti-
body to the cell surface using PAL as a fusion partner.
However, little positive binding to antigen was reported.
As the exposed portion of the PAL is relatively small, it is
possible that proteins and peptides are embedded in the
membrane and are not exposed properly. It is therefore
important to verify the successful display of proteins or
peptides on the cell surface by using substrates or
antibodies that cannot penetrate into the membrane.
However, these problems will probably be solved as new
ideas are implemented. One important strategy to solve
the above problems is optimizing the method for fusing the
carrier and passenger proteins. A spacer of appropriate
length can be used to permit correct folding of both carrier
and passenger proteins, and to prevent possible functional
interference betweenthe carrier and passenger or between
the passenger and the cell surface. Strauss and Gotz [42]
reported that the activity of surface-immobilized lipase
varied with the spacer length. The activity increased from
0.8 to 83 units per milligram lipase as the spacer length
varied from 10 to 92 amino acids. It seems that the length
of the cell-wall-spanning region of the carrier protein must
exceed a critical length to allow efficient folding of the
passenger protein. INP seems to be an excellent anchoring
motif because the number of its internal repeats can be
varied to give different spacer lengths (Fig. 2b).
Surface display of proteins consisting of more than one
subunit is a challenge. Surface display of several different
proteins and/or peptides is also a challenging goal. Use of
two or more different anchoring motifs, each displaying
different proteins, could be a solution. However, the dis-
play of multiple proteins might be a serious burden to the
cell, making cells grow poorly or perhaps even making
them die.
The development of a surface display system that is
robust enough for industrial applications is the ultimate
challenge. As described, many interesting surface display
systems have been developed for various applications but,
as yet, most (if not all) of these studies have been limited to
laboratory research. However, we believe that industrial
applications of cell-surface display will appear soon,
especially in the areas of bioconversion and peptide library
screening. As more new ideas are implemented to solve
problems such as difficulty of displaying large proteins or
multi-subunit proteins, even more successful commercial
applications of cell surface display will emerge.
Acknowledgements
Our work described in this paper was supported by the National Research
Laboratory program of the Ministry of Science and Technology, the Basic
Industrial Research Program of the Korean Ministry of Commerce,
Industry and Energy, and by the Center for Ultramicrochemical Process
Systems (CUPS).
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