Journal of Biotechnology 96 (2002) 129– 154

www.elsevier.com/locate/jbiotec

Review

Display of proteins on bacteria

Patrik Samuelson 1, Elin Gunneriusson, Per-A, ke Nygren, Stefan Ståhl *
Di6ision of Molecular Biotechnology, Department of Biotechnology, SCFAB, Royal Institute of Technology (KTH),
Roslagstullsbacken 21, SE-10691 Stockholm, Sweden
Received 23 July 2001; received in revised form 18 February 2002; accepted 6 March 2002

Abstract

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA
technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and
vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applica-
tions. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles
are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, ‘second
generation’ vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals,
through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby
increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with
heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether
bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of
tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal
ions can be created and potential environmental or biosensor applications for such recombinant bacteria as
biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries
for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired
properties can be selected from such libraries. This article explains the basic principles of the different bacterial
display systems, and discusses current uses and possible future trends of these emerging technologies. © 2002 Elsevier
Science B.V. All rights reserved.

Keywords: Surface display; Gram-negative bacteria; Gram-positive bacteria; Vaccine delivery; Microbial biocatalyst; Bioadsorbent;
Environmental application

Abbre6iations: aa, amino acid; AIDA-I, adhesin involved in the diffuse adhesion; CMCase, carboxymethylcellulase; CTB, cholera
toxin B subunit; CwbA, cell wall bound autolysin modifier protein; Fft, b-D fructosyltransferases; FnBPA, fibronectin binding
protein A; FnBPB, fibronectin binding protein B; HBsAg, hepatitis B surface antigen; HCV, hepatitis virus C; HIV, human
immunodeficiency virus; HPV, human papilloma virus; i.n., intra nasally; Inp, ice-nucleation protein; i.p., intra peritoneally; i.v.,
intra venously; Lpp, major lipoprotein; OmpA, outer membrane protein A; OmpS, outer membrane protein S; OPH, organophos-
phorus hydrolase; OprF, outer membrane protein F; OspA, outer surface protein A; PAL, peptidoglycan associated protein; PhoA,
alkaline phosphatase; RSV, respiratory syncytial virus; SLH, S-layer homology motifs; SPA, staphylococcal protein A; SPG,
streptococcal protein G; TTFC, tetanus toxin fragment C; vag, vaginally.
* Corresponding author. Tel.: + 46-8-5537-8329; fax: +46-8-5537-8481.
E-mail address: stefans@biochem.kth.se (S. Ståhl).
1
Present address: Department of Chemical Engineering, University of Texas, Austin, TX 78712-1095, USA.

0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 0 4 3 - 3
130 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
1. Introduction
Cell surface localized molecules is a common
theme in nature, and the processes governed by
different surface proteins are fundamental to
many biological phenomena, such as cell– cell
recognition, signal transduction, surface adher-
ence, colonization, immunoreactions, and others.
Protein –protein or protein– carbohydrate interac-
tions or bridging mechanisms constitute the domi-
nating interactions in these processes (Westerlund
and Korhonen, 1993). Scientists have utilized
some of these naturally occurring surface proteins
as carriers for foreign molecules to be displayed
on the surface of cells and viruses. This review
will focus on the molecular mechanisms used in
the various bacterial surface display systems, and
will in addition give an overview of the different
applications. For an extensive review with further
emphasis on the applications, see Benhar, 2001.
2. Bacterial surface display systems
The first reports about bacterial display of for-
eign peptides/proteins, genetically fused to carrier
proteins, were published in 1986 by Freudl and
co-workers (Freudl et al., 1986) and Charbit and
co-workers (Charbit et al., 1986), respectively.
Since then, a number of different bacterial surface
display systems have been developed and investi-
gated as, among other things, vaccine delivery
vehicles, ‘library and selection’ devices, cellular
adsorbents, and biocatalysts (Georgiou et al.,
1997; Ståhl and Uhlén, 1997; Benhar, 2001).
2.1. Systems for Gram-negati6e bacteria
Characteristic for Gram-negative bacteria is
that they have an inner cell membrane and a cell
envelope, the outer membrane, and in between a
peptidoglycan cell wall structure. The inner mem-
brane is a typical phospholipid bi-layer, while the
outer membrane consists of two leaflets of differ-
ent character. The inner leaflet is composed of
phospholipids and the outer leaflet mainly of lipo-
polysaccharides. For proteins to be targeted to the
extracellular medium or the outer membrane they
must, therefore, cross both the cytoplasmic and
the outer membrane. This can be accomplished by
different mechanisms depending on the type of
protein and its specific secretion pathway. For
extensive reviews regarding the different secretion
pathways, see Cheng and Schneewind (2000), Fer-
nandez and Berenguer (2000), Buchanan (2001),
Plano et al. (2001), Sandkvist (2001), van Wely et
al. (2001). In order to achieve surface exposure of
heterologous proteins on Gram-negative bacteria,
researchers have used naturally ‘secretion com-
petent’ proteins as carriers of the protein of inter-
est. This process has been shown to be critically
dependent on properties of the passenger, i.e. the
heterologous peptide or protein to be displayed,
as well as the carrier (Sandkvist and Bagdasarian,
1996). In the following section some of the sys-
tems used will be described, and for an overview
see Table 1.
2.1.1. Outer membrane protein-based systems
The outer membrane proteins span the mem-
brane several times, and differ from other mem-
brane spanning proteins in the sense that the
‘spanning structure’ is mainly anti-parallel b-
strands, making up b-barrel structures. A number
of different functions have been ascribed to them,
such as membrane traffic warden, phage recep-
tors, and others.
Outer membrane protein A (OmpA) from dif-
ferent Gram-negative bacteria has shown to be
highly homologous (Cole et al., 1982). Most of
the amino acid (aa) differences can be found in
the N-terminal part, and are clustered in four
regions (Morona et al., 1984, 1985) that have been
shown to constitute surface exposed loops (Cole
et al., 1983; Freudl et al., 1986; Nguyen et al.,
1998). The observed variabilities suggested that
these loops might be suitable to harbor foreign
peptide/protein fragments for surface exposure. In
1986, Freudl and co-workers introduced a gene
fragment, encoding 15 aa, into the fourth outer
loop of the Escherichia coli (E. coli )-ompA gene
and showed that it was surface exposed in an
accessible form when expressed in E. coli (Freudl
et al., 1986). This indeed showed that outer mem-
brane proteins could serve as carriers of het-
erologous gene products to be displayed at the
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154 131

outer surface of Gram-negative bacteria. Later it
was also shown that it is possible to use the second
and third loop, either individually or simulta-
neously, to display peptides/proteins of interest on
Gram-negative bacteria (Freudl, 1989) (Fig. 1). A
hybrid OmpA was described, consist-ing of parts of
the E. coli and the Shigella dysenteriae ompA genes
(Pistor and Hobom, 1988), that was able to accept
insertions in the third and fourth outer loop (Fig.
1), either individually or simultaneously, for sur-
face exposure in both E. coli and Salmonella
typhimurium (Schorr et al., 1991).

Table 1
Selected reports of Gram-negative surface display systems and the intended applications in the presented examples

Carrier Passenger size Applications References

Outer membrane proteins

OmpA 15–514 aa Vaccines Freudl et al., 1986; Schorr et al., 1991;
Haddad et al., 1995
OprF 17–43 aa Vaccines Wong et al., 1995
LamB 11–232 aa Vaccines, cellular adsorbents, peptide libraries Charbit et al., 1986; Brown, 1992, 1997;
Steidler et al., 1993
OmpS 38–115 aa Bacterial targeting, study protein–protein Lång and Korhonen, 1997; Lång et al.,
interactions, epitope mapping 2000
OmpC 162 aa Bioremediation Xu and Lee, 1999
PhoE 8–32 aa Vaccines Agterberg et al., 1987, 1990
Invasin 18 aa Peptide libraries Nakajima et al., 2000
Lpp%OmpA 540 kDa Biocatalysis, antibody libraries, Francisco et al., 1992, 1993b; Daugherty et
immobilization, bioremediation, biosensor al., 1998; Bae et al., 2000; Shi and Wen Su,
2001
Lipoproteins
TraT 11–98 aa Vaccine Taylor et al., 1990; Chang et al., 1999
PAL 250 aa ScFv antibodies, bioremediation Fuchs et al., 1991; Dhillon et al., 1999
OprI 16 aa Vaccine Cornelis et al., 1996
Inp 547 kDa Biocatalysis, vaccine, directed evolution Jung et al., 1998a; Kim and Yoo, 1999;
Kwak et al., 1999; Lee et al., 2000
Autotransporters
Igab 12 kDa Vaccine, bioremediation Klauser et al., 1990; Valls et al., 2000
VirGb 50 kDa Biocatalysis Suzuki et al., 1995
AIDA-I 12–40 kDa Vaccine, biocatalysis Maurer et al., 1997; Lattemann et al., 2000
Secreted
Pullulanase Biocatalysis Kornacker and Pugsley, 1990b
Subunits of surface appendages
Flagellae 11–115 aa Vaccines, bacterial targeting, peptide libraries, Kuwajima et al., 1988; Newton et al., 1989;
study protein–protein interactions Lu et al., 1995; Westerlund-Wikström et al.,
1997
Fimbriae 7–52 aa Vaccines, peptide libraries, bioremediation Hedegaard and Klemm, 1989; Jennings et
al., 1989; Thiry et al., 1989; Bakker et al.,
1990; van Die et al., 1990; Pallesen et al.,
1995; Schembri and Klemm, 1998; Rani et
al., 1999; Schembri et al., 1999; Kjaergaard
et al., 2000
S-layer proteins
RsaA 12 aa Not indicated Bingle et al., 1997

Note, vaccine application does not necessarily mean an oral or intranasal vaccine trial, but rather that the bacterium has been
evaluated as a live immunization vehicle.
132 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
Fig. 1. Schematic presentation of E. coli OmpA with a novel
epitope expressed in a surface-exposed loop. Rectangles repre-
sent membrane-spanning b-strands of OmpA.
LamB is an outer membrane protein that be-
longs to the porin class that can be found in E.
coli. It allows passive diffusion of small hy-
drophilic molecules and also takes up maltose and
maltodextrins specifically (Ferenci and Boos,
1980). A number of bacteriophages also use it as
a specific receptor (Thirion and Hofnung, 1972).
The active LamB consists of three 421 aa long
monomers (Clément and Hofnung, 1981). Each
subunit forms a b-barrel of 18 consecutive an-
tiparallel b-strands (Hofnung, 1995; Schirmer et
al., 1995) with protruding loops and turns on
either side of the outer membrane. These loops
and turns have been the subject for search of
‘permissive’ sites that can tolerate insertions with-
out loss of all biological properties of the carrier
and passenger proteins (Newton et al., 1996).
Several such permissive sites have been identified.
The first successfully used insertion sites for sur-
face display of foreign peptides were aa positions
153 (LamB153) and 374 (LamB374), respectively
(Charbit et al., 1986). When the 11 aa C3 epitope
from the VP1 coat protein of type 1 poliovirus
was inserted into these sites, it was shown that the
fusion protein could be expressed, directed and
correctly inserted into the outer membrane of E.
coli. It was also demonstrated that the C3 epitope
could be recognized, from the outside, with C3
specific antibodies in immunofluorescence as well
as immunogold electron microscopy analyses
(Charbit et al., 1986). However, although LamB
seems to be quite flexible regarding the properties
of the inserted sequences, it has been claimed to
have some restraints in the size of the insert
(Charbit et al., 1988). The upper size limit seems
to be 60–70 aa (Charbit et al., 1988; Su et al.,
1992), but one exception has been reported in
which a 232 aa staphylococcal protein A (SPA)
fragment was successfully displayed when using
LamB153 (Steidler et al., 1993). Amino acid 253,
as well as several other sites, has also been shown
to be a permissive surface exposed site (Charbit et
al., 1991; Newton et al., 1996).
OmpS is a 43 kDa outer membrane protein
from Vibrio cholerae that belongs to the LamB
glycoporin family. It is highly homologous to
LamB, both in terms of structure and function
(Lång and Korhonen, 1997). Like the other mem-
bers of this family it has nine loops facing the
exterior, that all might be potential targets as
carrier of passenger peptides. Lång and Korhonen
(1997) have displayed the 11 aa C3 epitope from
type 1 poliovirus, the 15 aa cholera toxin B sub-
unit (CTB) epitope CTP3, one, two and three
fibronectin binding D-repeats (38 aa) of FnBPA
of Staphylococcus aureus, and a 186 aa PapG
fragment (Lång et al., 2000) at the outer mem-
brane of E. coli and V. cholerae by introducing
the corresponding gene fragments into the fourth
loop of OmpS.
Like LamB, PhoE is an abundant outer mem-
brane protein that can be found in E. coli. It is
induced under phosphate limitations, and the ac-
tive form consists of trimers that build up mem-
brane channels. Through this pore small
hydrophilic molecules can pass in a diffusion-like
process (Nikaido and Vaara, 1985), and it has
also been shown that it functions as a receptor for
certain bacteriophages (Chai and Foulds, 1978).
The PhoE monomer traverses the outer mem-
brane 16 times in a b-strand configuration,
thereby exposing eight loops at the outer cell
surface. Since these loops have been shown to be
hypervariable (Mizuno et al., 1983; Van der Ley
et al., 1987) they constitute good candidates as
permissive sites. The fourth and fifth loop of
PhoE have successfully been used as carriers of
foreign peptides for display at the outer mem-
brane surface of E. coli (Agterberg et al., 1987,
1990) and S. typhimurium (Agterberg et al., 1988).
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
There has been indications that there are limita-
tions with respect to the size of the insert that can
be expressed using this system, since larger inserts
have resulted in less viable cells (Agterberg et al.,
1990).
A recent addition to the growing list of outer
membrane proteins that have been utilized for
heterologous surface display is the invasin protein
of Yersinia pseudotuberculosis. Y. pseudotuberculo-
sis invasin promotes bacterial entry by binding to
host cell integrins (Isberg et al., 1987; Isberg and
Leong, 1990). It is a 986-residue protein of which
the 500 N-terminal aa are thought to reside in
the outer membrane (Hamburger et al., 1999).
The C-terminal 497 residues, which make up the
extracellular part, are involved in the binding to
integrin (Leong et al., 1990). Nakajima and co-
workers developed a system for expression of
random peptides on the cell surface of E. coli by
creation of a fusion hybrid between a peptide and
the invasin protein (Nakajima et al., 2000). The
fusion protein consists of the first 625 aa of
invasin, a six aa long proline spacer, and a de-
camer of random peptides flanked by cysteine
residues. Through systematic screening of this ex-
pression library for its binding ability against
human cultured cells, the authors identified sev-
eral bacterial clones whose binding to human cells
was mediated by the surface displayed peptides
(Nakajima et al., 2000).
Generally, when outer membrane proteins are
used for display of heterologous peptides or
proteins, this is achieved through genetic insertion
into permissive sites of the carrier protein. This
means that the system becomes highly dependent
of the structural properties of the inserted protein
domain, since the peptide or protein will be more
constrained when inserted into a permissive site as
compared with when fused in the N- or C-termi-
nus of a protein. A system that combines the
benefits of efficient surface display of outer mem-
brane proteins and which allows C-terminal fu-
sions is the Lpp%OmpA system developed by
Georgiou and coworkers (Georgiou et al., 1996).
The major E. coli lipoprotein (Lpp) differs from
other outer membrane proteins in that all infor-
mation that is needed for targeting and insertion
into the outer membrane resides in the signal
133
sequence and the first nine N-terminal aa (collec-
tively denoted Lpp%). Fusions to the short Lpp%
sequence become fatty acylated, exported via the
lipoprotein pathway and inserted into the outer
membrane but are not surface exposed (Ghrayeb
and Inouye, 1984). By constructing a chimerae
consisting of: (i) the Lpp% region; (ii) aas 46–159
of OmpA (comprising transmembrane region B3–
B7); and (iii) the complete mature b-lactamase
sequence, Francisco and co-workers (Francisco et
al., 1992) managed to display b-lactamase in a
functional form on E. coli (Fig. 2). They have
since then used versions of this Lpp%OmpA-system
with different numbers of OmpA transmembrane
regions to investigate the efficacy as display vec-
tors in terms of display efficiency, outer mem-
brane integrity and protease stability. This
resulted in a version, able of surface display, that
takes advantage of only one transmembrane re-
gion (aa 46– 66) and which is suitable for C-termi-
nal fusions (Georgiou et al., 1996). The
Lpp%OmpA (aa 46– 159) system looks promising
since it is well suited as a carrier of relatively large
inserts, and it has been extensively used for the
display of enzymes, single chain antibody frag-
ments (scFv) and binding domains on E. coli
(Georgiou et al., 1997). However, a disadvantage
with the system is that it seems to be sensitive to
extensive secondary and tertiary structures of the
passenger (Stathopoulos et al., 1996).
Fig. 2. Schematic presentation of the expected structure of the
chimeric Lpp%OmpA construct in the outer membrane.
Rectangles represent membrane-spanning b-strands of OmpA.
134 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
2.1.2. Lipoproteins
This is a group of bacterial proteins that are
anchored to the outer membrane mainly via a
covalently attached lipid moiety, hence their name
lipoproteins. TraT is a surface-exposed,
oligomeric, plasmid-encoded lipoprotein. It con-
fers reduced ability of the host to act as a recipi-
ent in conjugation so called surface exclusion
(Achtman et al., 1977) and it also mediates serum
resistance (Moll et al., 1980). The mature form is
a 220 aa polypeptide that is anchored to the outer
membrane via an N-terminal covalently cysteine-
attached lipid moiety (Perumal and Minkley,
1984). The central section contains two hydropho-
bic regions that most likely span the outer mem-
brane. Taylor et al. (1990) introduced the C3
epitope from type 1 poliovirus into a number of
different positions in TraT, and found that aa
positions 61, 200 and 216 resulted in surface
display of the C3 epitope, when expressed in E.
coli. Recently, Chang and colleagues (Chang et
al., 1999) surface expressed various antigens (up
to 98 aa) as fusions to TraT. This system should
potentially result in high levels of surface expres-
sion since native TraT is represented in approxi-
mately 10 000 copies, and mutants with as high as
up to 200 000 copies have been reported (Man-
ning et al., 1982).
The peptidoglycan-associated lipoprotein
(PAL) from E. coli has been used as a carrier of
proteins for presentation at the surface of E. coli.
Fuchs et al. (1991, 1996) made a fusion of: (i) a
pectate lyase signal peptide; (ii) a single chain
antibody fragment; and (iii) the peptidoglycan
associated protein component of PAL, and
showed that this construct was capable of display-
ing active scFvs at the surface of E. coli, although
the membrane appeared to be leakier than nor-
mally. The result was quite surprising since the
lipid modification site in PAL had been removed.
Due to the lack of further reports on the PAL-
system, except from a surface displayed anti-
atrazine scFv (Dhillon et al., 1999), the general
applicability is not known.
Pseudomonas syringae has an ice-nucleation
protein (Inp) that normally resides on the surface
of cells. It is membrane anchored via the glycosyl-
phosphatidylinositol (GPI)-anchor sequence, and
this is quite unique for being a prokaryote since
this motif is normally found only in eukaryotic
cells (Ferguson and Williams, 1988; Schreuder et
al., 1996). By fusing levansucrase to the C-termi-
nus of Inp and expressing this construct in E. coli,
the recombinant bacteria obtained surface local-
ized enzyme activity (Jung et al., 1998a,b). In fact,
Inp is one of the more promising display systems
for Gram-negative bacteria available. It has
shown capable of displaying enzymes (Jung et al.,
1998a,b; Kim et al., 2000; Jeong et al., 2001;
Shimazu et al., 2001), single-chain antibodies
(Bassi et al., 2000) and antigens (Kim and Yoo,
1999; Lee et al., 1999).
2.1.3. Autotransporters
Autotransporters have the unique feature with
respect to their secretion mechanism that they
promote their own transport through the outer
membrane of Gram-negative bacteria. This is
done without any additional proteins needed be-
sides the ones involved in the Sec-dependent
translocation across the inner membrane. So far,
this type of proteins has only been associated with
Gram-negative pathogens where they play a role
in virulence. The first protein to be studied in
more detail was the IgA1 protease from Neisseria
gonorrhoeae (Pohlner et al., 1987), thereby giving
rise to the name of a novel family: IgA protease-
like autotransporter family. The members of this
class of secreted proteins have several structural
similarities. They all have three functional do-
mains: (i) an N-terminal signal peptide; (ii) a
domain to be secreted or surface displayed; and
(iii) a C-terminal autotransporter structure, mak-
ing up a membrane embedded b-barrel through
which the domain to be secreted/displayed is
translocated in an unfolded state (Jose et al.,
1995). The transport mechanism can be described
as follows. First, the N-terminal signal peptide
specifies the transport of the polypeptide across
the cytoplasmic membrane, most probably using
the Sec dependent pathway (Klauser et al., 1992),
then the C-terminal part associates with, and as-
sembles into the outer membrane as a b-barrel,
and finally the ‘passenger’ is translocated through
the channel formed by the b-barrel. In some cases,
as with IgA protease, the homologous ‘passenger’
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
protein is released and maturated through sequen-
tial autoproteolytic cleavages after translocation
(Pohlner et al., 1987).
Some of the IgA protease-like members have
been investigated as possible carriers of foreign
proteins to be surface displayed (Table 1). Al-
though, this class of proteins has been shown to
be capable of translocating relatively large passen-
gers, the process appears to be critically depen-
dent on the secondary and tertiary structures of
the target protein (Jose et al., 1995; Suzuki et al.,
1995).
The IgA protease is an extracellular protease,
from the Gram-negative pathogen N. gonor-
rhoeae, specific for human IgA1. It is produced as
a 169 kDa precursor that is processed in several
steps by signal peptidase and autoproteolytic
cleavage to form the mature released 106 kDa
IgA protease (Pohlner et al., 1987). The precursor
can be divided into four structural/functional do-
mains: (i) the N-terminal signal peptide; (ii) the
protease segment; (iii) a very basic a-domain; and
(iv) the b-domain (Igab) which holds the essential
outer membrane functions. The 45 kDa b-domain
consists of two regions, the membrane embedded
core (30 kDa) and the surface exposed linking
region (15 kDa) (Klauser et al., 1992). The core
region assembles in the outer membrane as a
channel through which the linking region and the
attached passenger protein, in this case the
protease, are translocated. By replacing the
protease domain in IgA protease with the 13 kDa
CTB Klauser et al. (1990) managed to display
CTB on the surface of E. coli. They also showed
that the translocation, which seems to take place
in a linear fashion, is critically dependent on the
conformation of the passenger (Klauser et al.,
1990, 1992, 1993).
The protein VirG of Shigella is essential for
bacterial spreading. It is responsible for the local-
ized deposition of filamentous actin in the cyto-
plasm of epithelial cells (Prevost et al., 1992). The
37 kDa C-terminal b-core of VirG has been pre-
dicted to form a channel, composed of 14
transmembrane amphiphatic b-sheets and one a-
helical transmembrane structure, in the outer
membrane (Suzuki et al., 1995). Through this
channel, the preceding 80 kDa N-terminal portion
135
is translocated and subsequently released, most
likely due to autoproteolytic cleavage. MalE and
PhoA fusions to the N-terminus of the 37 kDa
VirG b-core (VirGb) have been displayed on the
surface of E. coli (Suzuki et al., 1995).
AIDA-I is an E. coli adhesin involved in the
diffuse adhesion (AIDA-I) to mammalian epithe-
lial cells (Benz and Schmidt, 1989). After translo-
catation through the cytoplasmic membrane the
C-terminal autotransporter domain is postulated
to form a b-barrel of 14 amphiphatic b-sheets
within the outer membrane (Maurer et al., 1997).
A CTB-fragment and a five aa epitope (PEYFK)
have been successfully displayed at the surface of
E. coli by fusing them to the linking region of
AIDA-I (Maurer et al., 1997). This system was
shown to be more efficient than Igab and VirGb
for heterologous surface display (Maurer et al.,
1997). In addition, active b-lactamase was recently
displayed on E. coli using this system (Lattemann
et al., 2000).
2.1.4. Secreted proteins
Pullulanase is a maltose-inducible starch-de-
branching enzyme from Klebsiella pneumoniae.
The secretion is dependent on products from at
least eight secretion genes that are located on both
sides of the pullulanase gene (pulA) in the chro-
mosome of Klebsiella (Kornacker and Pugsley,
1990b). Pullulanase can be expressed and secreted
in E. coli provided simultaneous expression of the
pullulanase secretion genes (d’Enfert et al., 1987).
The protein exists as an exposed, cell surface
bound intermediate before being released to the
medium at the on-set of the stationary growth
phase (Michaelis et al., 1985). The export and
secretion information seem to reside in the N-ter-
minal half of pullulanase (Kornacker and Pugsley,
1990a). Hybrid proteins in which the C-terminal
region of pullulanase had been replaced by the
mature b-lactamase and alkaline phosphatase
(PhoA), respectively, thus fused to the first 832
N-terminal aa of pullulanase, were shown to be
transiently surface-anchored when expressed to-
gether with the pullulanase secretion genes in E.
coli (Kornacker and Pugsley, 1990b). Although,
both enzymes were displayed at the surface, PhoA
was less efficiently exposed, which might be due to
136 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
incompatible secondary and tertiary structures of
the heterologous passenger protein.
2.1.5. Subunits of surface appendages
Flagella are external filamentous structures that
are involved in the motility of bacteria. The flagel-
lum is composed of several thousand copies of the
FliC major protein, flagellin, as well as some
other accessory proteins (Wilson and Beveridge,
1993). Flagella can be divided into several anti-
genicity groups, conferred by the hypervariable
central region in flagellin (Joys, 1988; Wilson and
Beveridge, 1993). The N- and C-terminal regions
of flagellin are highly conserved among different
species while the central region shows a significant
variability (Wilson and Beveridge, 1993), suggest-
ing that this part of the flagellin is not essential
for filament structure. This was also shown to be
the case when Kuwajima et al. (1988) deleted a
large part (187 bp) of the hypervariable region of
E. coli fliC-gene with still retained flagellar poly-
merization and motility. Since then, the central
region of the fliC gene and other flagellin genes
has been the target position for insertions of
foreign peptide/protein gene sequences to be dis-
played on flagella. The first successful report
along these lines was the display of an eleven aa
epitope from hen egg-white lysozyme on the E.
coli flagellar filament (Kuwajima et al., 1988).
An interesting variant of flagellar display is the
FLITRX-system, described by Lu et al. (1995).
They constructed a fusion protein in which the
entire coding sequence of E. coli thioredoxin
(trxA) was inserted into the dispensable region of
flagellin gene, fliC. Into a disulfide-constrained
surface loop of the thioredoxin, they introduced a
random dodecapeptide library and displayed this
on flagella at the bacterial surface (Lu et al.,
1995).
The FLITRX-system may not be compatible
with the functional display of certain larger
proteins or protein fragments due to their intrinsic
structural requirements. On the other hand, this
system could be well suited for the display and
selection of peptides exhibiting affinity against
various molecular targets of interest. For such an
application, it is often advantageous to have the
peptides displayed in a constrained format, since
this generally leads to higher affinities. Moreover,
a constrained display format may allow grafting
of the peptide, with retained activity, into another
scaffold.
The versatility of these different flagella-based
systems is dependent on the size and conforma-
tion of the passenger structures, and there is a
debate regarding the size the flagellum can harbor
without severe disturbance of its biogenesis. Most
reports claim that the permissive size is less than
60 aa (Georgiou et al., 1997), but there have been
reports where a 302 aa YadA adhesin was ex-
pressed in the H7 flagellin (Westerlund-Wikström
et al., 1997; Tanskanen et al., 2000).
Fimbriae are long filamentous bacterial ad-
hesins that mediate bacterial targeting to and
colonization of specific host tissues. The fimbriae
consist of approximately 1000 copies of the major
subunit, fimbrillin, and also some minor protein
components that are important for the adhesion
and filament assembly, respectively. Bacterial cells
house up to about 500 copies each of these sur-
face organelles. The major subunit shows regions
of hypervariability that determine the fimbrial
antigenicity (for reviews see Krogfelt, 1991; Hult-
gren et al., 1993). The hypervariable regions have
been the main focus for insertion of foreign gene
products in fimbriae.
P fimbriae are common among many uropatho-
genic E. coli strains where they mediate the adher-
ence to uroepithelial cells. van Die et al. (1990)
have inserted epitopes in two (out of five) hyper-
variable regions, HR1 and HR4, of P fimbrillin
and it was shown that up to 14 aa could be
exposed without severe disturbance of fimbriae
biogenesis.
Type 1 fimbriae mediate the binding of E. coli
to epithelial receptors containing mannose
residues. Two different subunit types have been
used as carriers of foreign epitopes, namely the
major subunit, FimA, and one of the minor sub-
units, FimH. In an early experiment, three differ-
ent insertion points in FimA were identified that
all gave rise to almost normal fimbriae fenotype
when harboring small insertions (Hedegaard and
Klemm, 1989). Although most reports have sug-
gested an upper size-limit of approximately 25 aa
(Pallesen and Klemm, 1994), Stentebjerg and co-
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
workers recently managed to display a 34 aa
CTB-peptide when inserted in FimA (Stentebjerg-
Olesen et al., 1997). FimH is present as an integral
part of the fimbriae and is responsible for the
receptor binding, and it binds to D-mannosides.
FimH can be divided into two different functional
domains, the N-terminal receptor-binding domain
and the C-terminal transport recognition domain.
A 52 aa preS2 hepatitis B virus segment and a 15
aa CTB subunit peptide, CT3p, have been ex-
pressed within FimH at two different sites and the
chimeric fimbriae were successfully surface-ex-
posed and immunogenic (Pallesen et al., 1995).
For other types of fimbriae that have been used
in the context of surface display, see Table 1.
2.1.6. S-layer proteins
Although S-layers are being increasingly iden-
tified on Bacteria and Archaea, their function is in
most cases still unknown. In a few instances,
S-layers have been shown to be virulence factors
on pathogens, a depository for surface-exposed
enzymes, shape-determining agents, and nucle-
ation factors for fine-grain mineral development
(Beveridge et al., 1997). Yet, for the vast majority
of S-layered bacteria, the natural function of these
crystalline arrays continues to be evasive.
Caulobacter crescentus is a Gram-negative bac-
terium that produces a two-dimensional crys-
talline array on its surface composed of a single
98-kDa protein, RsaA. The RsaA protein inter-
locks in a hexagonal pattern to completely en-
velop the bacterium (Bingle et al., 1997). Bingle
and co-workers (Bingle et al., 1997) successfully
surface displayed a 12 aa peptide from Pseu-
domonas aeruginosa strain K pilin within the
paracrystalline S-layer of C. crescentus. This was
achieved by genetically inserting the correspond-
ing gene fragment into various positions of the
rsaA gene (Bingle et al., 1997).
2.2. Systems for Gram-positi6e bacteria
Compared with Gram-negative bacteria, Gram-
positive bacteria have a much thicker cell wall
that makes them more rigid (Fischetti et al.,
1996). Apart from this difference they also lack
the outer membrane envelope, that is characteris-
137
tic for the Gram-negative genera, and this absence
should theoretically simplify extracellular secre-
tion of heterologous proteins in Gram-positive
bacteria.
There are more than 100 known surface
proteins of Gram-positive bacteria (Ton-That et
al., 1997). Many of these proteins share some
conserved features needed for cell wall anchoring.
To be correctly translocated through the cellular
membrane and subsequently anchored to the cell
wall, the surface proteins should have an N-termi-
nal signal peptide and a C-terminal cell wall sort-
ing signal (Schneewind et al., 1992). The sorting
signal has been extensively studied and it consists
of: (i) a conserved pentapeptide motif, LPXTG;
(ii) a hydrophobic stretch of 15–22 aa; and (iii) a
short charged tail (6– 7 aa) (Fischetti et al., 1990;
Schneewind et al., 1993) making up a total of
approximately 35 aa. In S. aureus, the LPXTG
motif serves as the recognition sequence for prote-
olytic cleavage between its threonine and glycine
residues (Navarre and Schneewind, 1994), fol-
lowed by subsequent linkage of the threonine to a
branched peptide, via the amino of the penta-
glycine crossbridge, in the peptidoglycan layer
(Schneewind et al., 1995; Ton-That et al., 1997;
Navarre and Schneewind, 1999; Mazmanian et
al., 2000). To achieve surface display of het-
erologous proteins on Gram-positive bacteria, this
type of general cell wall sorting and anchoring
mechanism has been the most frequently utilized
strategy (Ståhl and Uhlén, 1997; Ståhl et al., 2000;
Hansson et al., 2001). However, as can be seen in
Table 2, other types of carrier proteins have also
been used.
2.2.1. Cell wall bound proteins
Protein A of S. aureus (SPA) is the best charac-
terized Gram-positive cell wall anchored protein.
It interacts with several specific host molecules
(for example immunoglobulins, kininogen and a2-
macroglobulin) during infection, resulting in ad-
hesion to host tissues and escape from the defense
system (Goward et al., 1993; Foster and McDe-
vitt, 1994). It consists of an N-terminal signal
peptide, five (in some strains four) IgG-binding
domains (Uhlén et al., 1984), a highly repetitive,
proline and glycine rich region, X, described to
138 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154

Table 2
Selected reports of Gram-positive surface display systems and their intended applications in the presented examples

Carrier Passenger size Applications References

Cell wall bound proteins

SPA 15–397 aa Vaccines, diagnostics, adsorbents, Samuelson et al., 1995; Hansson et al.,
biocatalysis, immobilization 1992; Strauss and Götz, 1996; Ståhl et al.,
1997; Steidler et al., 1998
M6 15–438 aa Vaccines Pozzi et al., 1992; Medaglini et al., 1995;
Fischetti et al., 1996; Di Fabio et al., 1998
FnBPA, FnBPB 263–397 aa Biocatalysis Strauss and Götz, 1996
SpaP1 179 aa Vaccines Lee et al., 1999
PrtP 52 kDa Vaccines Norton et al., 1996
Cell membrane anchored proteins
DppE 192 aa Vaccines Acheson et al., 1997
CwbA 192–497 aa Vaccines Acheson et al., 1997
Mtb19 31 kDa Vaccines Stover et al., 1993
Cell surface associated proteins
Fft No passenger Not indicated Rathsam et al., 1993
SLH 50 kDa Vaccine, biocatalysis Mesnage et al., 1999a,b

Note, vaccine application does not necessarily mean an oral or intranasal vaccine trial, but rather that the bacterium has been
evaluated as a live immunization vehicle.

span the cell wall (Guss et al., 1984), and finally
the C-terminal cell wall sorting region, here de-
noted M (Guss et al., 1984). The XM-region of
SPA has been used as a fusion partner together
with different spacers and various signal peptides
to achieve surface exposure of heterologous sur-
face proteins on S. xylosus (Hansson et al., 1992),
S. aureus (Schneewind et al., 1992), S. carnosus
(Samuelson et al., 1995) and Lactococcus lactis
(Steidler et al., 1998). As can be seen in Table 2,
this strategy has resulted in the display of target
proteins of various lengths, 15– 397 aa.
The M6 protein belongs to a group of a-helical
coiled-coil fibrillar proteins that can be found on
the surface of group A streptococci. These
proteins are responsible for the anti-phagocytic
property of the group A streptococci, and there
are more than 80 different serotypes. The particu-
lar M6 protein can be found in Streptococcus
pyogenes and it is a 53.5 kDa polypeptide with
the typical above outlined features needed for cell
wall anchoring. Pozzi and co-workers replaced a
538 bp long surface exposed region of M6 with a
297 bp epitope, E7, from human papilloma virus
type 16 and inserted this construct into the chro-
mosome of the commensal Streptococcus gordinii
(Pozzi et al., 1992). This strategy has led to the
successful exposure of foreign epitopes of various
lengths (15– 438 aa) at the surface of S. gordinii
(Di Fabio et al., 1998).
Piard et al. (1997) have expressed plasmid en-
coded wild type M6 protein and shown cell wall
anchoring in different heterologous hosts, such as
L. lactis, Lactobacillus fermentum, Lactobacillus
sake, and Streptococcus thermophilus. Although
this suggested a certain generality of the targeting
and surface attachment mechanism for this type
of proteins, it also revealed some differences in
anchoring efficiency among the bacteria tested
(Piard et al., 1997). The cocci, including S. pyoge-
nes, L. lactis, and S. thermophilus, all showed
detectable M6 protein in the supernatant, while in
a second group including the Lactobacillus species
L. sake and L. fermentum, anchoring appeared to
be complete.
Recently, Lee and co-workers (Lee et al., 1999)
inserted a gene fragment encoding the N-terminal
179 aa of the Bordetella pertussis S1 subunit into
the middle part of spaP1, the surface protein
antigen P1 from Streptococcus mutans. When this
gene construct was transformed into S gordinii the
expressed fusion protein was surface localized and
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
mainly associated with the cell wall (Lee et al.,
1999).
When L lactis and other lactic acid bacteria
grow in milk, they depend on the degradation and
release of aas from milk proteins. The initial
degradation in this process is performed by the
extracellular cell wall bound proteinase PrtP,
which also is involved in the cell separation pro-
cess (Buist et al., 1998). Norton et al. (1996) used
the C-terminal part of PrtP, that contain the
regular cell wall sorting signal of Gram-positive
bacteria, in an attempt to display the 52 kDa
tetanus toxin fragment C (TTFC) on L. lactis.
Interestingly, although the fusion protein was ex-
pressed in L. lactis it was not accessible at the cell
surface and was found in the membrane fraction,
instead of being correctly anchored in the cell
wall. This was likely due to that the TTFC was
not compatible with the membrane translocation.
By translational fusion of the collagen-binding
S-layer protein CbsA of Lactobacillus crispatus to
the cell wall sorting signal of PrtP, CbsA was
presented and functional at the surface of Lacto-
bacillus casei (Martinez et al., 2000). Perhaps the
CbsA-PrtP system may be functional as a carrier
for other polypeptides of interest.
2.2.2. Cell membrane anchored proteins
Bacillus subtilis has a lipoprotein, DppE (also
called DciAE), involved in the sporulation process
and transport of di-peptides (Mathiopoulos et al.,
1991). The entire coding sequence of dppE was
introduced as fused to the C-terminal 192 aa of an
invasin from Y. pseudotuberculosis and expressed
in B. subtilis (Acheson et al., 1997). This resulted
in expression of the fusion protein and targeting
to the membrane, but the fusion protein was not
surface-accessible unless the bacteria were
stripped from their cell wall. This is probably due
to ‘shielding’ by the thick peptidoglycan layer.
Also the cell wall bound autolysin modifier
protein (CwbA) of B. subtilis has been used as a
fusion partner to achieve surface exposed chimeric
proteins in Bacillus. Acheson et al. (1997) fused
the complete CwbA coding sequence to different
lengths of the invasin gene, which resulted in
exposition of the fusion protein containing 192 up
to 497 aa of the invasin.
139
In order to obtain surface exposure of a het-
erologous antigen, the outer surface protein A
(OspA) of Borrelia burgdorferi, on the Mycobac-
terium bo6is strain bacille Calmette-Guerin
(BCG), Stover et al. (1993) fused OspA to the
relevant N-terminal part of the Mycobacterium
tuberculosis lipoprotein Mtb19. The OspA was
found to be correctly surface exposed and the
recombinant BCG strain was further evaluated in
extensive vaccination studies (see also Section
3.1.1).
2.2.3. Cell surface associated proteins
The b-D fructosyltransferases (Ffts) of oral
streptococci constitute a family of proteins that
polymerize the fructose moiety into extracellular
fructans. Most of these enzymes are secreted into
the surroundings but the Fft of Streptococcus
sali6arius (SsFft) is initially cell-associated and
subsequently released upon exposure to its sub-
strate, sucrose. This mechanism appears to be
unique for SsFft (Milward and Jacques, 1990).
SsFft has many of the properties of a Gram-posi-
tive surface anchored protein, namely: (i) an N-
terminal signal peptide; (ii) a proline–
glycine–threonine–serine rich cell wall spanning
domain; (iii) a hydrophobic stretch; and (iv) a
charged tail, however, it lacks the LPXTG-motif.
The native Fft has been expressed in S. gordinii
and it exhibited wild type-SsFt functions in this
heterologous host (Rathsam et al., 1993). This
system may become useful for certain applications
as a carrier of heterologous proteins to be surface
exposed and subsequently released when desired.
Many surface proteins of Gram-positive bacte-
ria contain motifs, about 50 aa long, called S-
layer homology motifs (SLH). The display
mechanism involves a noncovalent interaction be-
tween the SLH domain and peptidoglycan-associ-
ated polymers (Mesnage et al., 2000). Bacillus
anthracis synthesizes two S-layer proteins, EA1
and Sap, each with three SLH motifs toward the
amino-terminus. By constructing chimeric genes
encoding the SLH domains fused to levansucrase
of B. subtilis (Mesnage et al., 1999a) or tetanus
toxin fragment (Mesnage et al., 1999b), the hybrid
proteins were stably expressed on the surface of B.
anthracis.
140 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
3. Surface display applications
The use of recombinant microorganisms with
surface exposed protein structures has during the
last decade gained a lot of interest since this type
of research holds great promise in different areas
such as biotechnology, microbiology and
biomedicine (Georgiou et al., 1997; Ståhl and
Uhlén, 1997; Hansson et al., 2001). Traditionally,
the by far most studied area of application has
been vaccine development. More recently also
biocatalysis, biosensor technology, bioremediation
and different library/selection technologies have
come into focus. Some examples of such applica-
tions and aspects thereof will be discussed.
3.1. Applications in immunology and 6accinology
3.1.1. Li6e bacterial 6accine deli6ery
In the context of vaccine development, live
delivery of subunit vaccines has been looked upon
as an attractive alternative to the more traditional
methods, the use of protein subunits and inacti-
vated agents. This is due to the ease of production
and also that they sometimes elicit long-lasting
immunity after only a single immunization
(Dertzbaugh, 1998). For the development of live
vaccine delivery vehicles, it has generally been
considered advantageous with cell surface dis-
played heterologous antigens for the induction of
antigen-specific antibody responses (Leclerc et al.,
1991; Stover et al., 1993; Haddad et al., 1995;
Nguyen et al., 1995; Georgiou et al., 1997; Titball
et al., 1997), but some disagreeing opinions have
been raised (Wick et al., 1993).
To generate live bacterial vaccines, two differ-
ent types of bacteria have been used. Either,
normally pathogenic bacteria that have been sub-
jected to attenuation, such as Gram-negative
Salmonella spp (Dertzbaugh, 1998) and the
Gram-positive M. bo6is strain BCG (Stover et al.,
1993, 1994; Langermann et al., 1994), or non-
pathogenic commensal or food-grade bacteria,
such as S gordinii and several staphylococcal and
lactic acid bacteria, respectively (reviewed in Fis-
chetti et al., 1996; Pozzi and Wells, 1997; Ståhl et
al., 1997; Mercenier, 1999). Non-pathogenic or
food-grade bacteria that do not invade the host
would generally be inefficient in generating strong
antibody responses. However, it has recently been
demonstrated that vaccine delivery systems based
on food grade bacteria can be significantly im-
proved by the co-display of adhesins that will
assist in the targeting to the mucosal epithelium
(Cano et al., 1999; Liljeqvist et al., 1999). Protec-
tive immunity to respiratory syncytial virus was in
fact evoked in mice using this strategy (Cano et
al., 2000). The major concern with the use of
BCG and Salmonella is the safety in infants and
immunocompromised individuals (Mekalanos,
1994). This concern is less pronounced in the
approach with commensal or food-grade bacteria.
However, since research is progressing, time will
show which approach is the most appropriate
regarding the efficacy and safety.
Although there is still some debate regarding
the necessity of surface display for the develop-
ment of live vaccine vehicles, the technology un-
doubtedly opens up some very interesting
possibilities for the future. These are for example,
simultaneous surface exposure of different anti-
gens and: (i) immunomodulating structures (spe-
cific T- or B-cell epitopes); (ii) receptor-specific
molecules for targeting to specific immunoreactive
sites; or (iii) colonization factors, in order to
enhance the potency of the vaccine and to recruit
desired immune responses. An overview present-
ing some of the used systems and the obtained
results can be found in Table 3.
3.1.2. Epitope mapping
Phage display of random peptide libraries has
been extensively utilized for epitope mapping
(Dunn, 1996; Smith and Petrenko, 1997). The
same methodology can be applied to other mi-
croorganisms and eukaryotic cells as well, how-
ever, this research field has just started to emerge.
Lu et al. (1995) presented an example, where they
displayed a random dodeca peptide library in the
surface loop of thioredoxin, inserted into the hy-
pervariable region of flagellin displayed on the E.
coli flagella. This system was capable of identify-
ing the epitope sequences for the three antibodies
tested.
Christmann and colleagues presented another
system suitable for display of constrained peptides
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154 141

Table 3
Selected examples where live bacteria with surface displayed antigens have been used as vaccine vehicles

Display system Organism Displayed antigen Animal model Results References

G-negati6e
OmpA S. typhimurium Malarial (SERP, Mice (orally) Ag. specific IgG and Schorr et al.,
HRPII) IgM 1991
Chimeric S. typhimurium Malarial (M3) Mice (i.p.) Ag. specific IgG Haddad et al.,
OmpA 1995
LamB E. coli HBsAg (preS2) Rabbits and mice Ag. specific IgG Charbit et al.,
(i.v.) 1987
S. typhimurium Polio virus type 1 Mice (i.p.) Ag. specific IgG and Leclerc et al.,
epitope (C3) IgM 1991
Flagellin (H1-d) S. dublin HBsAg Guinea pigs and Ag. specific IgG Wu et al., 1989
mice (orally)
CTP3-epitope from CTB Mice (i.p.) Ag. specific IgG Newton et al.,
1989
Influenza hem-agglutinin Rabbits (i.m.) and Ag. specific IgG and McEwen et al.,
epitope mice (orally) sIgA+partial protection 1992
HIV1-gp41-epitope Mice (i.p.) Ag. specific IgG Newton et al.,
1995
Inp S. typhi Ty21a HBsAg, HCV Mice (i.n. and i.p.) Ag. specific Lee et al., 2000
IgG+partial protection
G-positi6e
SPA S. xylosus RSV antigen Mice (orally) Ag. specific IgG Nguyen et al.,
1993
S. carnosus Streptococcal protein G Mice (orally) Ag. specific IgG Ståhl et al., 1997
(SPG)
S. carnosus SPG/CTB Mice (i.n.) Ag. specific IgG and Cano et al.,
IgA 1999
S. carnosus CTB/RSV Mice (i.n.) Protection Cano et al.,
2000
M6 S. gordinii HIV1 gp120 epitope Proliferation assay Th-proliferation Pozzi et al.,
1994
HPV epitope (E7) Mice (i.n.) Ag. specific IgG Oggioni et al.,
1995
Allergen from white-face Mice (orally and Ag. specific IgG and Medaglini et al.,
hornet venom (Ag5.2) i.n.) sIgA 1995
HPV epitope (E7) Mice (vag.) Ag. specific IgG and Medaglini et al.,
sIgA 1997
HIV1 epitope-V3 or Cynomolgus Ag. specific IgG and Di Fabio et al.,
HPV epitope-E7 monkeys (vag.) sIgA+T-cell 1998
proliferation
SpaP1 S. gordinii Pertussis toxin subunit Mice (i.p.) Protection Lee et al., 1999
S1
Autolysin mod. B. subtilis Invasin fragment Mice (orally) Ag. specific IgG Acheson et al.,
CwbA 1997
Lipoprot. M. bo6is-BCG OspA (Lyme borreliosis) Mice (i.p.) Ag. specific Stover et al.,
Mtb19 IgG+protection 1993
SLH B. anthracis TTFC Mice (subcut.) Protection Mesnage et al.,
1999a

aa, amino acid; CTB, cholera toxin B subunit; HBsAg, hepatitis B surface antigen; HCV, hepatitis virus C; HIV, human
immunodeficiency virus; HPV, human papilloma virus; i.n., intra nasally; i.p., intra peritoneally; i.v., intra venously; RSV,
respiratory syncytial virus; TTFC, tetanus toxin fragment C; vag., vaginally.
142 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154

Table 4
Selected examples of bacterially surface displayed enzymes

Display system Displayed enzyme Results Reference

Gram-negati6e
Lpp%OmpA b-lactamase Active enzyme anchored at surface Francisco et al., 1992
Cellulomonas fimi -¦- Francisco et al., 1993b
exoglucanase (Cex)
PhoA Inefficient display Stathopoulos et al.,
1996
OPH Active enzyme anchored at surface Richins et al., 1997
Inp Levansucrase -¦- Jung et al., 1998a
B. subtilis CMCase -¦- Jung et al., 1998b; Kim
et al., 2000
OPH -¦- Shimazu et al., 2001
AIDA-I b-lactamase -¦- Lattemann et al., 2000
pullulanse b-lactamase and PhoA Transient anchoring; b-lactamase actively displayed and Kornacker and
PhoA inefficient exposure Pugsley, 1990b
Gram-positi6e
FnBPB, SPA, S. hyicus lipase and Active enzymes anchored at surface Strauss and Götz, 1996
FnBPA b-lactamase
SLH Levansucrase -¦- Mesnage et al., 1999a

CMCase, carboxymethylcellulase; FnBPA, fibronectin binding protein A; FnBPB, fibronectin binding protein B; Inp, ice-nucleation
protein; OPH, organophosphorus hydrolase; PhoA, alkaline phosphatase; SLH, S-layer homology motifs; SPA, staphylococcal
protein A.

(Christmann et al., 1999). They used the cystine
knot of a squash-type protease inhibitor, fused to
Lpp%OmpA, as a structural scaffold for display of
conformationally constrained peptides on E. coli.
By combining magnetic cell sorting with fluores-
cence activated cell sorting (FACS), the authors
were able to enrich cells, displaying a particular
epitope of interest, 107-fold (Christmann et al.,
1999).
Lång and co-workers cloned various PapG frag-
ments into OmpS in order to characterize the
adhesive epitopes responsible for binding to glo-
boside (Lång et al., 2000). These techniques for
epitope mapping, constitute an attractive/comple-
ment alternative to the traditional synthetic pep-
tide-based techniques, and might find extensive use
in the future.
3.2. Display of enzymes
Biocatalysis is also a key area in biotechnology
(May, 1997). To perform an enzymatic reaction,
one can use either of two different strategies:
purified enzyme, free or immobilized or whole cells
expressing the enzyme of interest. The immobiliza-
tion can be performed in several different ways,
which can have both positive and negative effects
on the enzyme activity and stability (Scouten, 1995).
However, one obvious disadvantage with the first
strategy is the cost for enzyme purification. Obvi-
ously, the second approach eliminates the enzyme
purification, but it still has its drawback, namely,
the resistance to mass transport of the substrate and
product across the cell membranes if the enzyme is
expressed within the cell. A solution to the above
mentioned problems could be to, by recombinant
means, express the enzyme of interest as anchored
to the outer surface of a cell or bacteriophage. This
strategy would potentially eradicate the need for
enzyme purification, minimize the resistance to
mass transport and also result in a directed immo-
bilization, which might be beneficial.
Two early attempts along these lines were the
successful display of enzymatically active b-lacta-
mase, normally localized to the periplasmic space,
at the surface of E. coli using the pullulanase system
(Kornacker and Pugsley, 1990b) and the
Lpp%OmpA-system (Francisco et al., 1992), respec-
tively. Since then, several other, more relevant
enzymes and systems for surface display in Gram-
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
negative bacteria have been investigated (Table 4).
The general applicability of the Gram-negative
display systems, however, still need to be proven
since there will be certain enzymes, such as alka-
line phosphatase and various multimeric enzymes,
that may result in inefficient surface exposure
(Kornacker and Pugsley, 1990b; Stathopoulos et
al., 1996).
In a pioneering study, E. coli b-lactamase and a
lipase from S. hyicus were expressed on the outer
surface of the Gram-positive bacterium S. carno-
sus with retained activity (Strauss and Götz,
1996). Due to their rigid cell wall, Gram-positive
bacteria might constitute an attractive alternative
to Gram-negative bacteria as whole-cell catalysts
(Strauss and Götz, 1996). Another potential ad-
vantage is that there is only need for translocation
through one membrane, to achieve secretion and
subsequent surface exposure, in contrast to the
two membranes present in Gram-negative
bacteria.
A good practical example of what genetically
enzyme-coated bacteria can be used for is in
biosensor technology. Mulchandani et al.
(1998a,b) used recombinant E. coli with surface
expressed organophosphorus hydrolase in a
biosensor format for direct determination of
organophosphate nerve agents. The bacteria were
immobilized and connected to either a fiber-optic
bundle (Mulchandani et al., 1998a) or a potentio-
metric device (Mulchandani et al., 1998b) for
signal transduction. Both types of biosensors
showed good sensitivity and selectivity, and more-
over, they also showed very good stability when
used repeatedly more than 75 times.
Furthermore, large libraries of engineered en-
zymes have been displayed on the surface of
bacteria with the purpose of selecting enzyme
variants with novel substrate specificities (Olsen et
al., 2000) or improved substrate catalysis (Kim et
al., 2000). Based on differences in growth rates,
Kim and co-workers selectively screened for im-
proved variants of carboxymethyl cellulose (CM-
Case) displayed on E. coli, when the bacteria were
grown on CMCase plates (Kim et al., 2000). For
more on this type of applications, see Section 3.5.
143
3.3. Bacterial surface display for en6ironmental
applications
New interesting and promising biological
strategies for removal or detoxification of toxic
chemicals, e.g. heavy metals and xenobiotics, have
been presented (Lindow et al., 1989). These meth-
ods can be categorized as phytoremediation
(Chaney et al., 1997; Raskin et al., 1997; Rugh et
al., 1998) and bioremediation (Volesky and
Holan, 1995; Lovley and Coates, 1997), which are
based on the use of plants and microbes,
respectively.
For some bioremediation applications, it might
be beneficial to have the biologically active moiety
displayed at the outer surface of the microbe.
Recently, the first publications about the use of
various heavy metal-binding motifs displayed on
the surface of E. coli were reported (Sousa et al.,
1996, 1998; Mejàre et al., 1998; Schembri and
Klemm, 1998; Kotrba et al., 1999). When these
recombinant bacteria were tested for their ability
to survive in a Cd2 + -millieu the results were
somewhat contradictory. Sousa et al. (1996),
found that the E. coli had gained an enhanced
cadmium accumulation rather than an increased
metal tolerance, however, the results of Mejàre
and co-workers (Mejàre et al., 1998) stated the
opposite. Nevertheless, this new technique still
holds great promise, especially when having in
mind the possibility of expressing tailor-made,
highly specific, ligands (Klemba et al., 1995; Lu
and Valentine, 1997) for affinity capture of certain
pollutants. By engineering a mouse metalloth-
ionein on the cell surface of Ralstonia eutrophy
CH34 for immobilization of heavy metals in soil,
the toxic effects of the heavy metal on the growth
of tobacco plants were decreased significantly
(Valls et al., 2000). Recently, also Gram-positive
bacteria, such as staphylococci, have been investi-
gated in this context (Samuelson et al., 2000;
Lehtiö et al., 2001; Wernèrus et al., 2001).
The use of surface displayed enzymes for detox-
ification of toxic material has not yet come in use
to any large extent. However, Richins et al. (1997)
reported about the biodegradation of
organophosphorus pesticides by recombinant E.
coli, harboring surface expressed organophospho-
144 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
rus hydrolase. Since this type of application is still
in its infancy, a lot of research is still needed to
show a more general use of such approaches.
3.4. Surface display of antibody fragments and
6arious binding proteins
The possibility to display ligands, in the form of
peptides or protein fragments, at the outer surface
of microorganisms has opened up several poten-
tial applications. Some suggested applications are:
(i) studies on protein– protein interactions; (ii)
creation of whole-cell diagnostic tools; (iii) gener-
ation of devices for immunopurification; (iv)
whole-cell affinity sorbents; and (v) targeting cells
to specific imunoreactive sites or increase their
ability to colonize certain tissues (for more infor-
mation on the last vaccine related application, see
Section 3.1.1. Live bacterial vaccine delivery)
(Hofnung, 1991; Little et al., 1993; Francisco and
Georgiou, 1994; Georgiou et al., 1997).
For example, to characterize the binding inter-
actions between SPA and IgG, one, two or four
IgG-binding domains of SPA were fused to LamB
and surface expressed in E. coli, and subsequently
analyzed for IgG-binding activity in a whole-cell
format (Steidler et al., 1993). Lång and Korhonen
(1997) performed a similar investigation, in which
one, two or three fibronectin binding D-repeats of
FnBPA were expressed in OmpS at the surface of
E. coli. The cells, having surface exposed D-re-
peats had acquired fibronectin-binding activity as
determined by incubation of whole cells with ra-
dioactive fibronectin. In an interesting experiment
by Cho and co-workers (Cho et al., 1998), a yeast
cell surface display system was used for the dis-
covery of ligands that trigger cell activation. In
this report, yeast cells expressing high surface
levels of a T cell receptor ligand (a recombinant
antibody to the TCR Vb domain) were shown to
act as ‘pseudo’ antigen presenting cells and in-
duced T cell activation as monitored by increased
levels of CD25 and CD29 and by down regulation
of cell surface TCR. This indicated that such a
yeast display system, by virtue of its ability to
present ligands multivalently, might be used in
highly sensitive procedures to identify novel
polypeptides that interact in a multivalent manner
with cell surface receptors and thereby trigger
specific cellular responses (Cho et al., 1998). Such
applications could also be envisioned for many of
the other surface display systems presented in this
review.
In the field of antibody engineering, recent ad-
vances have greatly facilitated the recombinant
production of antibodies and antibody fragments
(Carter and Merchant, 1997; Hayden et al., 1997),
and we have just seen the beginning of this emerg-
ing field. One of the earliest examples of surface
expression of a single-chain Fv on a microbe, in
this case E. coli by taking advantage of the PAL-
system, was presented by Fuchs and co-workers
(Fuchs et al., 1991). Since then, some other
promising systems for antibody display in E. coli
(Francisco et al., 1993a), staphylococci (Gunner-
iusson et al., 1996) and yeast (Schreuder et al.,
1996; Boder and Wittrup, 1997) have been de-
scribed. Also other combinatorially engineered
binding proteins can be displayed on bacteria.
One such example is the display of S. aureus
protein A-based ‘affibodies’ engineered to bind
IgA or IgE, on the surface of S. carnosus cells
(Gunneriusson et al., 1999b). Similarly, a fungal
cellulose binding domain (CBD) was combinatori-
ally engineered to bind Ni2 + -ions, and when the
engineered CBDs were surface displayed on S.
carnosus cells, the recombinant bacteria were
found to have gained Ni2 + -binding (Wernèrus et
al., 2001). Such ‘cellular-antibodies’ could poten-
tially find use as simple whole-cell diagnostic
devices (Hofnung, 1991; Gunneriusson et al.,
1999a,b). Another potential alternative use could
be as whole-cell affinity chromatography matrices
for purification of interesting target proteins/
molecules. Chen et al. (1996) utilized E. coli cells
possessing surface-expressed scFv molecules in a
quantitative immunoassay that was reported to be
very rapid and accurate down to the nanomolar
level.
3.5. Bacterially displayed polypeptide libraries as
selection de6ices
The use of polypeptide/protein libraries and in
vitro selection technologies for the identification
and isolation of particular polypeptides with de-
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
sired traits (binding, catalysis, stability etc.) is a
rapidly growing field in bioscience. For construc-
tion of libraries at the genetic level, diversity can
be obtained by a variety of methods including for
example the use of natural gene pools such as
cDNA collections or immunoglobulin repertoires
from suitable donors, synthesis of oligonucle-
otides containing randomized stretches of nucle-
otides for use in PCR or direct cloning and
random mutagenesis by error prone PCR. These
and other methods have been extensively used to
generate libraries of for example antibody frag-
ments (Hoogenboom et al., 1998), peptides
(Cwirla et al., 1990; Devlin et al., 1990; Smith and
Petrenko, 1997) and protein ‘scaffolds’ (Nygren
and Uhlén, 1997; Skerra, 2000) from which par-
ticular members can be enriched and identified
using powerful selection technologies, including
systems based on surface display. The applied
selection pressure varies and is chosen on the
basis of the property sought for, most often bind-
ing (affinity).
A selection strategy offers a great advantage
over traditional screening procedures in that theo-
retically all variants (typically 105 – 109 species) in
the library are simultaneously subjected to a selec-
tion step that yields the ‘best’ molecules directly,
compared with a more tedious clone-by-clone
analysis format (Clackson and Wells, 1994).
Several different selection systems have been
described, such as phage display (reviewed by
Dunn, 1996; Smith and Petrenko, 1997; Hoogen-
boom et al., 1998), bacterial display (Georgiou et
al., 1997), yeast display (Boder and Wittrup, 1997;
Kieke et al., 1997), insect cell display (Ernst et al.,
1998), ribosomal display (Hanes and Plückthun,
1997), plasmid display (Cull et al., 1992; Schatz,
1993) and mRNA display (Wilson et al., 2001) (of
which only the bacterial display systems fall
within the scope for this review). The hall-mark of
all these systems is that the selected phenotype is
in some way physically linked to its genotype, for
example any selection procedure enriching for a
given mutant co-selects for its corresponding
DNA or RNA sequence (Clackson and Wells,
1994). Bacterial display might constitute an alter-
native to the dominating phage display technol-
ogy with some potentially beneficial features.
145
First, they are much simpler due to the use of
only one host, the bacterium, for propagation of
the library, compared with two, the bacteriophage
and bacterium, as in phage display. Second, there
is no need for reinfection in order to amplify the
selected variants. Third, the risk of affinity arti-
facts due to avidity effects, might be less pro-
nounced. Fourth, direct screening using FACS is
possible (Francisco et al., 1993a; Boder and Wit-
trup, 1997; Daugherty et al., 1998).
Combinatorial libraries displayed on the sur-
face of bacteria have been used for: (i) epitope
mapping, using random dodeca peptides in the E.
coli-FLITRX-system (Lu et al., 1995); (ii) isola-
tion of metal-recognizing peptides from a random
polypeptide library inserted into the maltodextrin
porin, LamB, of E. coli (Brown, 1997) or FimH
(Schembri et al., 1999; Kjaergaard et al., 2000);
(iii) antibody affinity maturation, taking advan-
tage of a Lpp%OmpA-displayed antibody library
subjected to fluorescence-activated cell sorting
(Daugherty et al., 1998, 2000); (iv) improvement
of enzyme catalysis (Kim et al., 2000); and (v)
changing the substrate specificity (Olsen et al.,
2000). The powerful screening methodology pro-
vided by FACS for the isolation of a rare recom-
binant microorganism, having a particular
heterologous protein exposed on its surface, was
demonstrated by Francisco et al. (1993a). They
utilized FACS to specifically enrich scFv-produc-
ing cells from a 105-fold excess of control cells in
only two steps. Recently, Olsen et al. created a
library of the protease OmpT, displayed on E.
coli. Screening a library of 6× 105 random OmpT
variants by FACS using a fluorescence energy
transfer (FRET) peptide substrate with a nonpre-
ferred Arg-Val cleavage sequence resulted in the
isolation of variant proteases with catalytic activi-
ties enhanced by as much as 60-fold (Olsen et al.,
2000).
The obtained results from the various cell sur-
face displayed libraries looks encouraging, how-
ever, one should be aware of its major limitations.
First, cell surfaces are much more complex than
those of bacteriophages, which could have nega-
tive effect on the molecular interactions of the
exposed polypeptide and its target are. The sec-
ond limitation is due to library size, since most of
146 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
the alternative hosts, except E. coli, generally re-
sult in less efficient transformation events. An-
other problem is the throughput rate of the
available FACS instrumentation, where the cur-
rent state of the art provides a maximum rate of
3.6 ×108 cells per h (Georgiou et al., 1997). De-
spite these limitations, cell surface displayed li-
braries, in combination with various technical
progresses, hold a great potential for the near
future.
4. Concluding remarks
As presented in this review, many systems exist
for surface display applications for both Gram-
negative and Gram-positive. From a practical
point of view, Gram-positive bacteria have certain
properties that potentially make them more suit-
able for bacterial surface display applications.
First, the surface proteins of Gram-positive bacte-
ria seem to be more permissive for the insertion of
extended sequences of foreign proteins, as com-
pared with the different Gram-negative surface
proteins (Fischetti et al., 1996; Ståhl and Uhlén,
1997; Hansson et al., 2001). For Gram-positive
bacteria, most systems for surface display rely on
a common mechanism for surface anchoring,
which allows insertion of heterologous protein
regions of several hundreds of aas. In contrast,
commonly used Gram-negative outer membrane
proteins that are surface anchored via multiple
passages through the outer membrane have only
the surface loops as ‘permissive sites’ for insertion
of foreign sequences and normally, though with a
few exceptions, allow only much shorter inser-
tions. Nevertheless, several, more recently devel-
oped systems, including the Lpp%OmpA system
and Inp system, allow insertion and surface dis-
play of larger proteins on E. coli cells (Georgiou
et al., 1997; Jung et al., 1998a,b; Benhar, 2001). A
second, more obvious, advantage with the Gram-
positive systems is that translocation through only
a single membrane is required to achieve proper
surface exposure of the heterologous polypeptide,
while in the Gram-negative systems both translo-
cation through the cytoplasmic membrane and
correct integration into the outer membrane are
required for surface display. Finally, considering
the practical handling of the bacteria, Gram-posi-
tive bacteria have the additional advantage of
being more rigid, due to the thicker cell wall
(Pagán et al., 1999), which thus allows various
laboratory procedures without extensive cell lysis.
One potential drawback for the Gram-positive
bacteria is their lower frequency of transforma-
tion, as compared with the Gram-negative bacte-
ria, a factor that is of obvious importance for the
creation of large combinatorial protein libraries.
Furthermore, certain Gram-positive bacteria such
as B. subtilis are known to excrete large amounts
of proteases, which could cause problem for sur-
face display applications. In contrast, S. carnosus
is described to have very low extracellular prote-
olytic activity (Götz, 1990). One advantage for the
Gram-negative bacteria such as E. coli and
Salmonella is that they are well studied organ-
isms. This means that there is a broad knowledge
about the genetic and biomolecular systems of
these bacteria, and this knowledge may become
useful in order to control the surface display in
certain applications.
Heterologous antigens have been surface dis-
played, and various bacteria have been extensively
investigated as vaccine vehicles for mucosal im-
munizations. As has been discussed here, their
capacity to induce potent antibody responses can
be significantly improved by co-display of ad-
hesins aimed for targeting to the mucosal epithe-
lium. Enzymes have been surface expressed in
approaches to create inexpensive whole-cell bio-
catalysts, and furthermore, libraries of enzyme
variants have been surface displayed for the pur-
pose of selecting catalysts with novel substrate
specificities or variants exhibiting improved catal-
ysis. Functional metal-binding peptides and
proteins have been surface displayed for environ-
mental and biosensor applications. In this con-
text, combinatorial approaches have also been
investigated to select peptides with improved se-
lectivity for certain metals. Single-chain antibody
fragments and other tailor-made binding proteins,
such as the SPA-based affibodies (Nord et al.,
1997; Gunneriusson et al., 1999a), have been ex-
posed on bacterial cell surfaces in attempts to
create bacteria with specific binding abilities for
 P. Samuelson et al. / Journal of Biotechnology 96 (2002) 129–154
use in various medical and biotechnology applica-
tions. In conclusion, it is evident that bacterial
display technology will be a continuously growing
research area in applied microbiology, vaccinol-
ogy and biotechnology. Different Gram-negative
and Gram-positive bacteria of various kinds will
most certainly be extensively investigated in dif-
ferent applications in the future.
Acknowledgements
P.S. would like to thank the Wenner-Gren
Foundations for financial support.
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