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Microbial 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 displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-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 allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-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 allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-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.
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 efciency 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 rst 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 lamentous 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-specic antibody responses [3,4]; screening-displayed peptide libraries by sequential binding and elution or, more efciently, by uorescence- 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 [811]; 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 [1619] 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 efciency 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 efcient 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 specic applications. For example, bacterial mbriae, S-layer proteins, ice nucleation pro- tein (INP) and some outer membrane proteins (such as Escherichia coli TraT) are efcient 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 inuences the immobilization efciency, stability, specic activity and post-translational modication 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 http://tibtec.trends.com 0167-7799/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(02)00006-9 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 efcient, 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 prole 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 inuence the trans- location process and nal 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 signicantly. The folding structure of the passenger protein (such as the formation of disulde 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 inefcient secretion in bacteria. Passenger protein con- taining four phenylalanine residues could not be displayed on the cell surface because of inefcient 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 efciency 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 disulde 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 Review TRENDS in Biotechnology Vol.21 No.1 January 2003 46 http://tibtec.trends.com 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 modied 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 lamentous 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 LppOmpA 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 efciency 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 Specic adhesin-receptor interaction [29] CcmA (5.1 kDa) Staphylokinase (SAK) (136 aa) Proteus mirabilis Studies Abbreviations: Adx, Bovine adrenodoxin; CABS, continuous afnity-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, bronectin binding protein A; GFP, green uorescent 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. Review TRENDS in Biotechnology Vol.21 No.1 January 2003 47 http://tibtec.trends.com good example of this type. An LppOmpA chimera consists of the signal sequence and rst nine N-terminal residues of the mature E. coli lipoprotein, and the residues 46159 or 4666 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 modication, 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 G F D L Q S K G K N L G R G Y D D E D I L K N K N L L D D N N Q F TR D A G I N T D T Y N A T R V G SL G WA N K Q N F A D A N N S K R T D A Q N TAA Y I G N G D R A E N K G Q Y Q V A F N L G D E G F G D A D G G N P S G E F TSGV T N NG R L R Q N G G Y R N T D F V G Y N R G R V A F A G L K F Q D V G Q I Q Y E W Q L G D V K G Y L D L K V G G S I T Y D Y S S I A G G I T Y T G G L K Y Q T Y Q A A L Y I E A V A Q Y Q F Y A L S P R L Y V D V G A T Y Y F N I K Y D V Y T S M I V A L G L V Y Q F Y M R L G F K G E T Q V V Y D V SW T T D V L P E F G G D T Y G S D N F M Q Q R G N G F A T G N S A E N E N N S W T H Y F S D N K D V D G D Q T N G D K N Y V E A T D Q L T G Y G S F D Y F G L V OUT IN Outer Membrane L1 L2 L3 L4 L5 L6 L8 L7 LQVDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLD PSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLDPSGHHHHHHSGLEDILQSKG 162 aa 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), LppOmpA (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 efciently exposed on the E. coli cell surface. Review TRENDS in Biotechnology Vol.21 No.1 January 2003 48 http://tibtec.trends.com 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 agella and mbriae (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 agella and mbriae 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 specic C-terminal sorting signal consisting of 3238 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 anking 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 http://tibtec.trends.com 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 uorescent protein (GFP) and blue uorescent protein (BFP) [14,41]. Some newly identied 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 uorescent protein; HBsAg, hepatitis B virus surface antigen; GFP, green uorescent 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 modicationprocess. Aga2p is linked to Aga1pby twodisulde 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 bronectin 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 afnity 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, bronectin 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 efcient 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 difculty 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). References 1 Smith, G.P. 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