The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

Andreas Christmann1, Kerstin Walter1, Alexander Wentzel, Ralph Krätzner2 and Harald Kolmar3

Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany 1 These authors contributed equally to this work


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel ß-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'–OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning ß-strand from residues 46–66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'–OmpA'–EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 107-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.

Keywords: combinatorial libraries/cystine knot/EETI-II/FACS/squash inhibitor/surface display


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In recent years, peptide libraries, presented on replicating entities via fusion to surface-exposed coat proteins, have been extensively used to discover novel peptide reagents with high affinity for molecular targets (Clackson and Wells, 1994Go; Scott and Craig, 1994Go; Lu et al., 1995Go; Yu and Smith, 1996Go). Although large repertoires of linear peptides have been successfully screened for high-affinity binders, imposing conformational constraints on the peptide molecules can confer a number of advantages that overcome at least some of the shortcomings of unstructured peptides (Clackson and Wells, 1994Go; Koivunen et al.,1995Go; Lu et al.,1995Go). Cyclic peptide libraries have been obtained by introducing cysteine residues flanking the variegated peptide sequence, thereby allowing loop closure via disulfide bond formation (Luzzago et al., 1993Go; McLafferty et al., 1993Go; Koivunen et al., 1995Go). Alternatively, the randomized sequence has been introduced into surface loops of highly constrained small proteins such as tendamistat (McConnell and Hoess, 1995Go), human pancreatic secretory trypsin inhibitor (Röttgen and Collins, 1995Go), bovine pancreas trypsin inhibitor (Roberts et al., 1992Go; Jespers et al., 1995Go) and other members of the Kunitz family of protease inhibitors (Dennis and Lazarus, 1994Go; Jespers et al., 1995Go; Markland et al., 1996Go). These proteins consist of about 55–75 amino acid residues and are stabilized by several intramolecular disulfide bonds.

The smallest proteins among the proteinaceous protease inhibitors, the squash family, are composed of only about 30 amino acids and have a very compact and rigid structure made up of a triple-stranded anti-parallel ß-sheet, which is held together by three disulfide bonds that give rise to a cystine knot (Pallaghy et al., 1994Go; Isaacs, 1995Go; Tamaoki et al., 1998Go). This knot consists of a ring formed by the first two disulfide bonds (1–4 and 2–5) and the intervening polypeptide backbone, through which the third disulfide bond passes (3–6) (Figure 1Go). The cystine knot structural motif appears to define one of the smallest stable domains and was identified in inhibitors from various sources acting against larger proteins like proteases, ion channels or protein receptors (Le-Nguyen et al., 1990Go; Pallaghy et al., 1994Go; Lin and Nussinov, 1995Go).



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Fig. 1. MOLSCRIPT (Kraulis, 1991Go) plot of the Ecballium elaterium trypsin inhibitor II structure, a member of the squash family of protease inhibitors, with the ß-strands represented by arrows and the disulfide bridges in black. The amino acid sequence and disulfide bond connectivity are shown under the schematic drawing.

 
A structurally and functionally well characterized member of the squash inhibitor family is the Ecballium elaterium trypsin inhibitor II (EETI-II) (Favel et al., 1989Go; Heitz et al., 1989Go; Le-Nguyen et al., 1990Go). Large quantities of EETI-II have been produced by chemical synthesis to allow structure determination by 2-D NMR (Heitz et al., 1989Go). The major features of the EETI-II secondary structure are a short 310-helix for sequence 11–15, a ß-turn 16–19 and a triple antiparallel ß-sheet 20–28 with a ß-turn formed by residues 22–25 (Figure 1Go). The inhibitor loop of EETI-II ranging from residues 3–8 is flanked by cysteine residues 2 and 9 and anchored through disulfide bonds to the structural framework.

Alterations of molecular recognition resulting in the inhibition of proteases other than trypsin have been achieved successfully by replacement of one or two residues in the EETI-II inhibitor loop (Le-Nguyen et al., 1990Go). This finding prompted us to explore the potential of the EETI-II cystine knot motif to provide a stable framework for the display of conformationally constrained peptides of various length and sequence, which are completely different from the wild-type sequence, aimed at presenting active residues for specific binding interactions. In this paper, we describe the construction of two variants of the squash inhibitor EETI-II, where the six-residue inhibitor loop is replaced by a 13- and 17-residue epitope sequence. We provide experimental evidence that these variations in loop sequence and length are well tolerated by the cystine knot structural framework.

Phage display has been exploited extensively to screen conformationally constrained peptide libraries for high-affinity binders by selective enrichment via binding of the surface exposed peptide to the protein target attached to a solid phase (Clackson and Wells, 1994Go). In recent years, combinatorial library presentation on the surface of bacterial cells has emerged as a potential alternative to phage display (Francisco and Georgiou, 1994Go; Georgiou et al., 1997Go). The power of this technology mainly lies in the ability to use fluorescence-activated cell sorting (FACS) for high-throughput screening of individual variants, which cannot be applied for enrichment of phage, since phage particles are too small to be detected by flow cytometer optics. Here, we report that the cystine knot protein EETI-II and the derived variants can be transported across the Escherichia coli outer membrane and anchored on the bacterial cell surface by fusion to the N-terminal targeting sequence of a Lpp'–OmpA' fragment. Epitope-presenting cells can be enriched against a large background of negative cells by antibody fluorescent labeling and using magnetic cell sorting combined with high-speed fluorescence-activated cell sorting.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Production of EETI-II variants with altered loop sequences

The starting point for the present study was a series of synthetic genes encoding EETI-II and two variants. EETI-II is composed of 28 amino acids. Residues 3–8 form the inhibitor loop with Arg4 being the reactive site P1 residue (Figure 1Go). To investigate whether the cystine knot (CK) of EETI-II is capable of presenting loop sequences that differ in length and amino acid sequence from the inhibitor loop, two EETI-II derivatives were constructed, where the six-residue inhibitor loop is replaced by a 13- and 17-residue linear epitope sequence (Figure 2AGo). EETI–CKSend contains a 13 amino acid sequence, which encompasses a linear epitope of Sendai virus L protein (Einberger et al., 1990Go). In EETI–CKEtag, which comprises an epitope from human bone Gla-protein (Kiefer et al., 1990Go) which is specifically bound by a monoclonal anti-E antibody (Pope et al., 1996Go), a 17-residue loop was placed between Cys2 and Cys9.



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Fig. 2. (A) Schematic representation of the expression vector pMX-EETI harboring the structural genes of MalE and the respective EETI variants. fdT, transcription termination sequence from bacteriophage fd; f1-IG, f1 replication origin; cat, chloramphenicol resistance marker. (B) Nucleotide and amino acid sequence at the junction of the malE–eeti-II gene fusion. Residues belonging to EETI-II are depicted in bold. (C) Amino acid sequence of EETI-II, EETI–CKSend and EETI–CKEtag. Invariable residues of the EETI-II framework are indicated in shaded boxes.

 
For wild-type EETI-II and EETI–CK variant expression in E.coli, vector pMX-EETI (Figure 2BGo) was constructed, where the synthetic genes are fused in frame to the malE gene encoding E.coli maltose-binding protein. A number of disulfide bond-containing proteins have been successfully produced via fusion to MalE (Maina et al., 1988Go), since the amino-terminal MalE moiety directs the fusion to the periplasmic space, where oxidizing conditions for disulfide bond formation prevail (Bardwell, 1994Go). After IPTG induction of malE–EETI transcription, cells were harvested and the fusion proteins were purified from the osmotic shock fluid of the bacterial cells by immobilized metal ion adsorption chromatography (Figure 3AGo). Yields of purified fusion protein were in the range 10–20 mg per liter of bacterial liquid culture. SDS–PAGE under non-reducing conditions showed the presence of the respective monomeric MalE–EETI fusion protein and, to a small extent additional dimers and higher oligomers (Figure 3AGo). Since these oligomers are absent upon addition of dithiothreitol as reducing agent, they are most likely formed by monomer concatenation through intermolecular disulfide bonds.



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Fig. 3. (A) 12.5% SDS–PAGE and (B) Western blot analysis of MalE–EETI fusion proteins. Panel 1 incubated with HRP-trypsin, panel 2 with anti-Sendai antibody and HRP-coupled anti-mouse antibody and panel 3 with anti-E antibody and HRP-coupled anti-mouse antibody. M, molecular mass markers with sizes indicated; w.t., EETI-II wild-type protein.

 
The purified MalE–EETI fusion proteins were blotted on to nitrocellulose followed by incubation with horseradish peroxidase-coupled trypsin, anti-Sendai or anti-E antibody. While both the monomer and the higher oligomers of MalE–EETI–CKSend and MalE–EETI–CKEtag were detected with the respective antibody, only the monomeric form of MalE–EETI-II wild-type was capable of binding trypsin. This indicates proper formation of the three intramolecular disulfide bonds and presentation of the trypsin inhibitor loop on the cystine knot scaffold. Removal of any of the three disulfide bonds in EETI-II by cysteine to serine replacement (C2S, C21S, C27S) completely abolishes trypsin inhibition (Wentzel et al., 1999Go). Accordingly, oligomeric forms of MalE–EETI-II, which are held together by intermolecular disulfide bonds, did not show any detectable trypsin-binding activity. The inhibitor constant for MalE–EETI-II towards trypsin was 9x10–9 M (Wentzel et al., 1999Go), which is considerably higher than that for natural EETI-II (1.2x10–12 M) (Le-Nguyen et al., 1990Go). Most likely, the amino- and carboxy-terminal extensions contribute to the reduced affinity for trypsin, since a chemically synthesized and in vitro refolded EETI-II with a C-terminal extension exhibits a similar inhibition constant to the MalE–EETI-II fusion protein (1.8x10–9 M) (Le-Nguyen et al., 1990Go).

The amount of free sulfhydryls in the three MalE–EETI fusions was quantitated by disulfide exchange with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman, 1959Go). No free cysteine sulfhydryls were detected (data not shown), supporting the notion that the cysteine residues of both the monomers of wild-type EETI-II and the variants EETI–CKSend and EETI–CKEtag are completely oxidized by intramolecular disulfide bond formation. This was confirmed by reversed-phase HPLC analysis of the EETI–CKSend protein, which was released from the MalE moiety of the fusion protein by IgA–protease cleavage (Kolmar et al., 1992Go). EETI–CKSend eluted as a single peak earlier than the respective fully reduced form (data not shown), which is indicative of the presence of a unique molecular species compacted by intramolecular disulfide bonds (Le-Nguyen et al., 1993Go). Although being highly unlikely (Wentzel et al., 1999Go), we cannot definitely rule out the possibility that this species contains disulfide-bonded cysteine pairs that are different from wild-type EETI-II. Taken together, these data indicate that the cystine knot fold of EETI-II is nearly quantitatively formed in high yield in vivo under the oxidizing conditions of the E.coli periplasm and that scaffold formation is compatible with the presentation of a loop sequence as long as 17 amino acid residues.

E.coli cell surface display

Recently, an Lpp'–OmpA' vehicle was reported that can target proteins fused to its carboxy terminus to the surface of the E.coli outer membrane (Francisco et al., 1992Go; Georgiou et al., 1996Go). This chimera consists of the signal sequence and the first nine amino acids of mature E.coli lipoprotein Lpp followed by amino acids 46–66 of the major outer membrane protein OmpA. According to the OmpA structure (Pautsch and Schulz, 1998Go), residues 46–66 comprise the third transmembrane ß-strand leaving its carboxy terminus exposed on the exterior of the cell. We have constructed the surface display vector pASK21–EETI which carries the gene encoding Lpp'–OmpA'–EETI-II under tetA promoter/operator control (Figure 4Go). Tight regulation is achieved by presence of the structural gene for the tet repressor on the same plasmid. Expression of the tripartite fusion protein can conveniently be induced by adding anhydrotetracycline at a low concentration (Skerra, 1994Go).



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Fig. 4. (A) Schematic drawing of surface display vector pASK21-EETI. This plasmid contains the tripartite lpp'–ompA21–eeti-II gene fusion under tetA promoter control (Skerra 1994Go). The structural gene for tet repressor (tetR) is located on the same vector together with an ampicillin (bla) and chloramphenicol (cat) resistance marker. (B) Nucleotide and amino acid sequence of the gene fusion encoding Lpp'–OmpA'–EETI-II. Residues belonging to Lpp', OmpA' or EETI-II are depicted in bold; intervening sequences are linkers, encode an IgA protease cleavage site (PPTP) or are defined by nucleotide sequences of restriction sites used for gene fusion construction. In pASK21T, the lpp' codon 9 (CAG) is replaced by a TAG amber codon.

 
E.coli strain BMH71-18 containing pASK21-EETI had a growth arrest after anhydrotetracycline induction (Figure 5Go). The same finding was made by Georgiou and co-workers when overexpressing in high yield a tripartite Lpp'–OmpA(46–159)–ß-lactamase fusion under lpp/lac promoter control (Francisco et al., 1993aGo; Georgiou et al., 1996Go; Daugherty et al., 1998Go). Overproduction of outer membrane-linked proteins often results in changes in the structure of the outer membrane and, as a consequence, in periplasmic enzyme leakage and cell death (Georgiou et al., 1997Go). To avoid growth arrest and cell rupture, we reduced the rate of production of the tripartite fusion protein by replacing the first glutamine codon in the Lpp'–OmpA'–EETI encoding sequence with an amber stop codon (Figure 4BGo). BMH71-18 contains a glutaminyl amber suppressor tRNA, which by recognition of the TAG codon gives rise to translational readthrough. Owing to the reduced efficiency of in vivo nonsense suppression compared with the translation of a CAG codon at the same position (Miller and Albertini, 1983Go), yields of the encoded protein are concurrently diminished. Expression of the lpp'–ompA'–eeti gene fusion containing an amber codon at position 9 of the lpp' gene in suppressor strain BMH71-18 had only a minor influence on cell growth and viability (Figure 5Go). To investigate whether the tripartite fusion protein encoded by pASK21T-EETI-II can be detected in the outer membrane of BMH71-18, proteins of the outer membrane fraction were blotted on to nitrocellulose and probed with horseradish peroxidase-coupled trypsin. A band migrating at the expected apparent molecular weight for the tripartite fusion was detected, confirming the presence of EETI-II in functional form in the E.coli outer membrane (data not shown).



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Fig. 5. Growth kinetics of BMH71-18 harboring pASK21-EETI-II or pASK21T-EETI-II. ({blacksquare}) pASK21-EETI-II; (•) pASK21T-EETI-II. Open symbols represent uninduced cells and closed symbols induced cells with anhydrotetracycline added at time point 0 min.

 
Detection by FACS

BMH71-18 containing either pASK21T-EETI–CKSend or pASK21T-EETI–CKEtag, respectively, was grown at 37°C and induced at an OD600 of 0.2 with 0.2 µg/ml anhydrotetracycline. After 30 min of induction, cells were washed and incubated with anti-Sendai or anti-E antibody, respectively. After washing, cells were incubated with biotinylated anti-mouse antibody followed by labeling with streptavidin–R-phycoerythrin conjugate. As judged by fluorescence microscopy, all cells were fluorescently labeled by this procedure. When cells of BMH71-18/pASK21T-EETI–CKSend presenting the Sendai virus epitope were incubated with anti-E antibody and vice versa, none of the cells became fluorescently labeled (data not shown). The fluorescently labeled cells were analyzed using a MOFLO flow cytometer (Figure 6Go). The fluorescence intensity of EETI–CKSend and EETI–CKEtag presenting cells labeled with the corresponding monoclonal antibody was clearly distinguishable from background cellular autofluorescence.



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Fig. 6. FACS histograms of recombinant BMH71-18 to determine cell surface presentation of the various EETI-II derivatives. (A) BMH71-18 harboring pASK21T-EETI–CKSend or pASK21T-EETI–CKEtag. Cells were successively incubated with mouse monoclonal anti-Sendai antibody, biotinylated anti-mouse antibody and streptavidin–R-phycoerythrin conjugate. (B) Same cells as in (A) but successively incubated with mouse monoclonal anti-E antibody, biotinylated anti-mouse antibody and streptavidin–R-phycoerythrin conjugate.

 
Enrichment of cells displaying EETI–CKEtag

In BMH71-18 containing pASK21T-EETI, a reduction in growth rate compared with uninduced cells was observed after about 100 min of induction (Figure 5AGo). To avoid cell death resulting from overproduction of the Lpp'–OmpA'–EETI fusion upon prolonged induction, cells were induced by anhydrotetracycline addition for 30 or 40 min, depending on the experiment. In order to estimate the percentage of viable cells that can be recovered from a sorting experiment, cells harboring pASK21T-EETI–CKSend were induced for 30 min, labeled as described above and sorted by FACS using a sorting gate ranging from 15 to 10 000 relative fluorescence units. Single cells were spotted with the single cell deposition unit of the MoFlo sorter (Cytomation) into the wells of a 96-well microtiter plate, prefilled with rich medium. After overnight incubation, wells were scored for bacterial growth and compared with uninduced cells, where the sorting gate was set around the maximum of autofluorescence. Forty-four turbid wells were counted for the induced cells and 73 for the uninduced cells, which indicates that viability of EETI–CKSend presenting cells is only slightly reduced.

To explore whether cells presenting a particular constrained peptide can be enriched by FACS from a large background of negative cells, EETI–CKEtag and EETI–CKSend presenting cells were mixed at a ratio of 1:107. Then 1.6x109 cells were subjected to labeling with anti-E antibody, followed by biotinylated anti-mouse antibody and streptavidin-coupled colloidal superparamagnetic microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). After washing, cells were finally incubated with streptavidin–R-phycoerythrin conjugate. Prior to FACS, labeled cells were presorted by magnetic separation (Miltenyi and Pflüger, 1992Go). This was achieved by passage of the cell population through a high gradient magnetic separation column (Miltenyi Biotech), and 1.5x106 cells eluted after removal of the magnetic field. Since the colloidal magnetic particles are too small to be detected by the flow cytometer, cells could be immediately subjected to FACS and were run through the MoFlow cell sorter at an event rate of 30 000 s–1 (Figure 7BGo) and sorted on the basis of fluorescence intensity. A sorting gate was chosen such that <0.5% of the control cells fell within the positive window. After immediate resorting, the collected cells were grown overnight in fresh medium, labeled and analyzed (Figure 7CGo); 8.6% of the total cell population were found in the positive fraction. Then 2x107 cells were subjected to two more consecutive rounds of FACS and transferred into fresh medium for overnight culture. Labeling with anti-E antibody of the induced cells from the overnight culture revealed that 38.9% of the cell population fell within the positive window (Figure 7DGo). Twenty individual cultures derived from single cells of the last sorting round were probed for the presence of the EETI–CKEtag coding sequence by PCR amplification of the EETI–CK gene. Of these, 13 contained the EETI–CKEtag and seven the EETI–CKSend coding sequence (data not shown), confirming the enrichment of EETI–CKEtag expressing cells from a 1:107 mixture after one round of magnetic cell sorting followed by four rounds of fluorescence-activated cell sorting.



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Fig. 7. Histogram data from enrichment of BMH71-18 containing pASK21T-EETI–CKEtag. The bar in each graph represents the sorting gate defined as a positive event. The percentage of cells counted as positive events is indicated. (A) A 107:1 mixture of BMH71-18/pASK21T-EETI–CKSend and BMH71-18/pASK21T-EETI–CKEtag prior to the first sorting round. (B) Mixture after MACS sort. (C) Mixture from round (B) after two consecutive runs of FACS and overnight cell growth. (D) Mixture from round (C) after two consecutive runs of FACS and cell growth.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study, peptides were displayed on the surface of E.coli cells within the structural context of the Ecballium elaterium squash inhibitor. EETI-II is composed of only 28 amino acid residues and is thus one of the smallest peptidic protease inhibitors known. The tertiary fold of EETI-II, a prototypic squash family inhibitor, is mainly stabilized by three intramolecular disulfide bonds where the Cys15–Cys27 bridge penetrates the macrocycle formed by the two other disulfide bridges together with the linking peptide sequence (Figure 1Go).

Folding of EETI-II, which has been studied in vitro with a chemically synthesized peptide, was found to be a clean and quantitative process (Le-Nguyen et al., 1989Go). This was attributed to the high propensity of the GPNG sequence connecting ß-strands 2 and 3 to form a ß-turn, which may facilitate association of ß-strands in early folding stages of EETI-II followed by covalent fixation of the tertiary fold by disulfide bond formation (Le-Nguyen et al., 1989Go, 1993Go). No attempts have been reported so far to produce squash inhibitors in functional form via bacterial gene expression and folding in vivo. Cytoplasmic protein production of squash inhibitors resulted in the deposition of insoluble and misfolded proteins in inclusion bodies (Chen et al., 1992Go; Kojima et al., 1996Go). We obtained EETI-II in functional form via fusion to E.coli maltose-binding protein, which directs the fusion protein to the periplasmic space. Oxidative conditions in the periplasm allow the formation of correctly folded and fully functional EETI-II in high yields (Figure 2Go). We intentionally chose MalE as the amino-terminal fusion partner for EETI-II production and translocation across the cytoplasmic membrane since this molecule does not contain any cysteine residues in its primary sequence that might interfere with EETI-II disulfide bond formation.

We replaced the EETI-II inhibitor loop, which is composed of six amino acid residues, by two epitope sequences, 13 and 17 residues in length. Yields of monomeric, completely disulfide-connected fusion proteins were similar to that of wild-type EETI-II. These data suggest that the EETI-II inhibitor loop is a permissive location for the introduction of a variety of peptide sequences and that these insertions do not interfere with folding of the cystine knot structural scaffold. This is not unexpected, since during the folding of EETI-II, formation of a rigid core which contains two native disulfide bonds [9–21, 15–27] precedes inhibitor loop anchoring. This core, which comprises residues 9–28, is the direct precursor of the natural, fully oxidized product and structurally closely related to it. The final step in the folding process is the fixation of the protruding amino-terminal inhibitor loop sequence on to the antiparallel ß-sheet framework by [2–19] disulfide bond formation (Le-Nguyen et al., 1993Go). This pathway is rather different from the folding of Kunitz domain protease inhibitors such as BPTI, which have been used extensively as structural scaffolds for phage display of constrained peptides (Roberts et al., 1992Go; Dennis and Lazarus, 1994Go; Jespers et al., 1995Go; Markland et al., 1996Go). In BPTI, which also contains three intramolecular disulfide bonds, the major accumulated folding intermediate is not the direct precursor of the natural product, but has to isomerize through isomers with non-native disulfide bridges to the direct precursor (Goldenberg, 1992Go). These differences in the folding pathway may account for the high propensity of EETI variants to form the native structure both in vitro and in vivo, irrespective of the nature of the N-terminal tail encompassing the inhibitor loop.

Recently, another protein with cystine knot-like tertiary fold, the cellulose-binding domain (CBD) of the fungal enzyme cellobiohydrolase from T.reesei was used as a structural template for production of a combinatorial library that has been successfully screened by phage display for variants with novel binding properties (Smith et al., 1998Go). CBD contains only the central two pairs of cysteine residues, but lacks the pair of cysteines, which in EETI-II tethers the inhibitor loop at its amino-terminus to the structural core. Since the amino acid residues in CBD which are responsible for binding to cellulose are not organized in a continuous loop sequence, but are distributed over the flat face of the molecule, variation was introduced by randomizing several residues that are located in two regions of the polypeptide chain (Smith et al., 1998Go). The fact that these residues are constituents of the structural framework, and therefore may be folding determinants, may impose some constraints on their variability. Nevertheless, these and our results suggest that the cystine knot scaffold appears to be a useful architecture for the generation of small inhibitor molecules with novel binding characteristics.

A number of methods have been developed for the display of foreign peptides and proteins on the surface of Gram-negative bacteria (Georgiou et al., 1997Go). Surface display requires passage across a cell envelope consisting of two membranes, the cytoplasmic and outer membrane, which are separated by the periplasmic space. Georgiou and co-workers pioneered the utilization of truncated forms of the major outer membrane protein OmpA for surface display of various foreign proteins such as ß-lactamase (Francisco et al., 1992Go), scFv fragments (Francisco et al., 1993aGo; Daugherty et al., 1998Go), cellulase and cellulose binding domains (Francisco et al., 1993bGo). Two variants, encompassing residues 46–66 and 46–159, spanning one or five of the eight OmpA transmembrane ß-strands, respectively (Pautsch and Schulz, 1998Go), have been used successfully as membrane anchors. We opted for the minimal OmpA(46–66) version fused to the signal sequence plus the first nine residues of E.coli lipoprotein Lpp (Georgiou et al., 1996Go) to promote transport through and anchoring on the outer membrane of the EETI-II cystine knot protein and derived variants. High-level overproduction of proteins fused to Lpp'–OmpA' was described as leading to growth defects and destabilization of the integrity of the outer membrane (Francisco et al., 1993aGo; Georgiou et al., 1996Go; Daugherty et al., 1998Go). To obviate cell lysis, the fermentation temperature was reduced to 25°C and low-level lpp'–ompA' gene expression resulting from the leakiness of the lpp/lac promoter was found to strike a balance between cell viability and protein accumulation on the surface of the bacterial cell (Georgiou et al., 1996Go, Daugherty et al., 1998Go). We used the tightly regulated tetA promoter/operator (Lutz and Bujard, 1997Go) to control Lpp'–OmpA'–EETI-II fusion protein production. To reduce the net accumulation of the fusion protein to a tolerable level, translational efficiency was diminished by introducing an amber codon into the lpp' codon sequence and usage of an E.coli amber suppressor strain. FACS analysis of antibody-labeled cells presenting EETI-II variants that contain the respective epitope sequence confirmed that expression levels are sufficient to discriminate between epitope presenting cells and background autofluorescence while cell viability is only slightly compromised.

Isolation of conformationally constrained peptides binding to a particular molecular target via bacterial surface display requires the screening of a relatively large number of presenting cells. For instance, complete randomization of the six-residue inhibitor loop in EETI-II using an NN(G/C) codon coding scheme would necessitate the generation and screening of at least 1x109 individual transformants. To evaluate the feasibility of using bacterial surface display for the enrichment of desired cystine knot peptides against a very large background, we devised a model experiment by mixing cells presenting EETI–CKSend and EETI–CKEtag at a ratio of 107:1 in a total of 1.6x109 cells. In the first round of enrichment, we combined magnetic cell sorting (MACS) with fluorescence-activated cell sorting aimed at cutting down the flow cytometer sorting time. Cells were labeled with anti-E antibody and biotinylated anti-mouse antibody, followed by consecutive incubation with streptavidin-coupled super paramagnetic colloidal microbeads and streptavidin–R-phycoerythrin conjugate. Owing to steric exclusion, only a fraction of the cell surface coupled biotin molecules is bound by the streptavidin magnetobeads, thus allowing direct subsequent fluorescence staining with streptavidin–R-phycoerythrin conjugate. Presorting with MACS led to enrichment of E-epitope containing cells and thereby to a 1000-fold reduction in the total cell number to an amount that can conveniently be screened by FACS. Only four rounds of sorting were required to achieve an enrichment of EETI–CKEtag presenting cells from a ratio of 1:107 to ~1:1.

In conclusion, magnetic cell sorting in combination with fluorescence-activated cell sorting applying event rates of up to 30 000 s–1 opens up the possibility of screening very large molecular repertoires with over 1010 initial bacterial cells in a short time. Hence high-throughput screening by cell surface presentation of molecular repertoires of conformationally constrained peptides, which are tethered by amino- and carboxy-terminal disulfide bonds to the EETI-II cystine-knot scaffold, may become a powerful technology complementary to phage display.


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 Abstract
 Introduction
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 Materials and methods
 References
 
Strains and reagents

E.coli strains BMH71-18 [F' lacIq lacZ{Delta}M15, proA+B+; {Delta}(lac–proAB)supE, thi] and WK6 [F' lacIq lacZ{Delta}M15, proA+B+; {Delta}(lac–proAB), galE, strA, nalr] were used for all cloning and protein production experiments. Restriction enzymes and DNA-modifying enzymes were purchased from MBI Fermentas and New England BioLabs. Tfl polymerase was obtained from Promega. Biotinylated trypsin and biotinylated rabbit anti-mouse antibody were obtained from Sigma. The monoclonal antibody (VII-E-7) against a 13-residue C-terminal epitope of Sendai virus L-protein (DGSLGDIEPYDSS) (Einberger et al., 1990Go) was a gift from H.Einberger and H.P.W.Hofschneider (Max-Planck-Institut für Biochemie, Martinsried bei München, Germany). Anti-E monoclonal antibody was obtained from Pharmacia Biotech. Streptavidin–R-phycoerythrin conjugate was purchased from Molecular Probes. All other chemicals were of analytical grade and obtained from Sigma.

Synthetic oligonucleotides

Synthetic oligonucleotides were purchased from NAPS (Göttingen, Germany) and MWG Biotech (Ebersberg, Germany). AC656: 5'-GACCGAGATAGGGTTGAGTG-3'; AW-pMBamHI-X: 5'-GCTCGGTACCCGGCCGGGGCTCCATCGAGGGTAGG-3'; EETI-II-up2: 5'-CCTAGGCCTCCAACGCCCGGGTGCCCGCGAATTCTGATGCGTTGCAAACAGGACTCCGAC-3'; EETI-II-lo2: 5'-CGGGGATCCGCAGAAACCGTTGGGCCCGCAAACGCAGCCAGCCAGGCAGTCGGAGTCCTGTTTG-3'; EETI-II-6His: 5'-CCCTCTAGAGCGGCCGCTTAATGGTGATGGTGATGGTGCGGGGACCCGCAGAAACCG-3'; EETI-L6N: 5'-GTTTGCAACGCATTANGATTCGCGGGC-3' (N = G, A, T, C); ETI-SEND: 5'-GTCCTGTTTGCATGATGAATCGTATGGTTCGATATCGCCTAAGCTTCCATCGCACCCGGGC-3'; ETI-ETAG: 5'-ACGCCCGGGTGCGGTGCGCCGGTACCGTATCCAGATCCGCTGGAACCGCGTGCCGCTTCTGGCTGCAAACAGGACTCCGAC-3'; IMG546: 5'-GCGCTCTAGATTACCAATGCTTA-3'; lppOmpAup: 5'-GCGCTCTAGATAACGAGGGCAAAAAATGAAAGCTACTAAACTG-3'; lppup: 5'-ACGTGGCGCCTCACTTTATCTAAGATGA-3'; Omp21-TAG: 5'-TAAAATCGATTAGGGAATTAACCCGTA-3'; RSPX-Xho: 5'-GCGCCTCGAGTGAATTTCGACCTCTAG-3'.

Microbiological procedures

The medium used for growth and maintenance of E.coli strains was dYT (1% yeast extract, 1.6% Bacto tryptone, 0.5% NaCl). Anhydrotetracycline (Acros, Greel, Belgium) was added to liquid media at a final concentration of 0.2 µg/ml. Chloramphenicol was used at a final concentration of 25 µg/ml.

DNA procedures

Standard DNA procedures, such as plasmid isolation, ligation, restriction analysis of plasmids and isolation of DNA fragments, were carried out as described by Sambrook et al. (1989). Site-directed mutagenesis was performed as described by Kunkel et al. (1987). Polymerase chain reaction (PCR) using Tfl polymerase was as follows: 30 s denaturation at 94°C, 30 s annealing at 53°C and 30 s elongation at 72°C, 30 cycles.

Plasmid constructions

The EETI-II gene was designed by choosing codons that allow the introduction of appropriate restriction sites and synthesized by annealing oligonucleotides EETI-II-up2 and EETI-II-lo2 which are complementary at their 3' ends followed by fill-in reaction using T4-DNA polymerase. To simplify the purification of the EETI-II protein, six additional histidine codons were introduced at the EETI-II gene 3'-terminus by PCR using the primer pair EETI-II-up2 and EETI-II-6His. The resulting PCR product was digested with StuI and EcoRI and ligated to vector pHKREI (Kolmar et al., 1992Go), which was digested with the same restriction enzymes to yield pHK-EETI. Nucleotide sequence analysis of the resulting EETI-II insert led to the identification of an undesired base exchange at codon 6 which results in a leucine to proline codon substitution. The correct loop sequence was introduced by directed mutagenesis (Kunkel et al., 1987Go) using the mutagenic oligonucleotide EETI-L6N which encodes an isoleucine, leucine or valine codon at position 6 of the EETI-II gene. An EETI-II gene encoding the desired leucine codon at position 6 was identified by nucleotide sequence analysis.

pHKMalE was constructed by ligating a 1.6 kbp EheI/XbaI fragment from vector pMal-p (Maina et al., 1988Go) encompassing the malE gene under tac promoter control into an EheI/XbaI-digested pHKREI (Kolmar et al., 1992Go). The EETI-II gene was ligated as a StuI/XbaI fragment into the similarly digested pHKMalE and hitherto placed in frame to the 3' end of the malE gene to give vector pM-EETI. For removal of the BamHI restriction site 5' of the EETI-II gene, the EETI-II gene was re-amplified by PCR using the primers AW-pMBamHI-X and AC656 and re-introduced as a KpnI/XbaI fragment into similarly digested pM-EETI. The resulting vector was named pMX-EETI-II. The nucleotide sequence encoding the 13-residue epitope from Sendai virus L protein was introduced by directed mutagenesis (Kunkel et al., 1987Go) using the mutagenic primer ETI-SEND. pMX-EETI–CKEtag was constructed by PCR amplification of the EETI-II gene using the primers ETI-ETAG and AC656 followed by cleavage with AvaI/BamHI and ligation into AvaI/BamHI-digested pMX-EETI-II.

The Lpp'–OmpA'–EETI-II encoding sequence was assembled in expression vector pASK75 (Skerra, 1994Go). To this end, the lpp'–OmpA'–ß-lactamase encoding sequence was amplified by PCR from vector pTX21 (Georgiou et al., 1996Go) using the primer pair lppup and IMG546. The resulting DNA fragment was cleaved with EheI/PstI and ligated into similarly digested pHK-EETI. The ß-lactamase encoding sequence preceding the EETI-II gene was removed by cleavage with EcoRI and StuI, followed by conversion to blunt ends using T4-DNA polymerase and religation. To remove the coding sequence for the six carboxy-terminal histidines, the resulting vector was digested with BamHI and XbaI, followed by conversion to blunt ends using T4-DNA polymerase and religation. The lpp'–OmpA'–EETI-II coding sequence was amplified by PCR using the primers lppOmpAup and RSPX-Xho. The resulting XbaI/XhoI-cleaved fragment was ligated into similarly digested pASK75 (Skerra, 1994Go). Finally, the resulting vector was linearized with SpeI. The 1.1 kbp BssHII fragment encoding the chloramphenicol resistance gene (cat) was isolated from pHKREI (Kolmar et al., 1992Go) and, after conversion to blunt ends by fill-in reaction of vector and fragment, ligated to pASK–lpp'–ompA'–EETI-II. The resulting construct was named pASK21-EETI-II. The amber codon 9 of lpp' was introduced by PCR amplification of the OmpA'–EETI-II coding sequence using the primer Omp21-TAG and RSPX-Xho. The OmpA'–EETI-II coding sequence was removed from plasmid pASK21-EETI-II by ClaI/XhoI digestion and replaced by the similarly digested PCR product containing the amber codon. Introduction of the amber codon was confirmed by nucleotide sequence analysis. The resulting construct was named pASK21T-EETI-II. To obtain pASK21-CKSend, pASK21T-CKSend and pASK21T-CKEtag, the respective genes residing in pMX-EETI–CKSend or pMX-EETI–CKEtag, respectively were obtained by SmaI/BamHI cleavage and ligated into similarly digested pASK21-EETI-II or pASK21T-EETI-II, respectively.

Production and purification of MalE–EETI fusion proteins

E.coli strain WK6, transformed with the respective pMX-EETI plasmid, was grown overnight in 1 l of dYT medium supplemented with 1 mM IPTG and chloramphenicol (25 µg/ml). Soluble periplasmic proteins were isolated essentially as described (Kolmar et al., 1992Go). Proteins were precipitated from the osmotic shock fluid by addition of ammonium sulfate to 80% saturation followed by 20 min of centrifugation at 12 000 g. The ammonium sulfate precipitate was dissolved in 20 ml of 50 mM sodium phosphate, 50 mM sodium chloride, pH 8.0, and applied to a column containing 4 ml of Ni-charged chelating Sepharose (Pharmacia Biotech). The column was developed under gravity flow and stepwise eluted with 10 ml of 50 mM phosphate buffer, pH 8.0, 50 mM sodium chloride containing 20, 40, 80, 100, 200 and 300 mM imidazole. The fraction containing 200 mM imidazole was dialyzed against 5 l of phosphate buffer, 50 mM sodium chloride, pH 8.0. Protein concentrations were determined from the A280 (Pace et al., 1995Go).

Horseradish peroxidase coupling to trypsin

Conjugation of periodate-oxidized horseradish peroxidase (HRP) to trypsin was performed as described (Hermanson, 1996Go), with modifications. A 4 mg amount of horseradish peroxidase was dissolved in 200 µl of sodium phosphate buffer, 0.15 M NaCl, pH 7.2. After addition of 20 µl of 0.088 M sodium periodate and incubation for 20 min in the dark, the oxidized enzyme was purified by gel filtration using an NAP5 column of Sephadex G-25 (Pharmacia Biotech), which was pre-equilibrated with 50 mM sodium borate buffer, pH 9.1. After addition of 1 mg of trypsin to the HRP-containing fractions, the sample was incubated for 2 h at room temperature. The HRP–trypsin complex was purified by gel filtration using an NAP5 column of Sephadex G-25 (Pharmacia Biotech), pre-equilibrated with 10 mM Tris–HCl, pH 8.0, 100 mM NaCl. For reductive amination, 10 µl of 5 M sodium cyanoborohydride (Sigma) prepared in 1 M NaOH per milliliter of reaction solution were added to the protein-containing fractions. After incubation for 30 min at room temperature, unreacted aldehyde sites were blocked by addition of 50 µl of 1 M ethanolamine, pH 9.6, per milliliter of reaction solution. Finally, the HRP conjugate was purified by gel filtration using an NAP5 column of Sephadex G-25 (Pharmacia Biotech), which was pre-equilibrated with 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. Samples were stored in aliquots at –70°C.

Western blotting

To the purified MalE–EETI fusion proteins or outer membrane proteins isolated by Sarkosyl extraction (Filip et al., 1973Go), 1 volume of sample buffer [8 M urea, 200 mM Tris-base, 2% (w/v) SDS, 15 mM bromophenol blue] was added. Protein samples were subjected to SDS–PAGE. Proteins were electroblotted on to Protran nitrocellulose (Schleicher and Schüll, Dassel, Germany). Blots were incubated with 1:1000 diluted stock solutions (1 mg/ml) of HRP–trypsin conjugate, anti-Sendai antibody or anti-E antibody. After incubation with the first antibody, blots were washed and incubated with HRP-conjugated rabbit anti-mouse immunoglobulins (Amersham Life Science), 1:1000 diluted. Blots were developed in 50 mM Tris–HCl, pH 7.5, containing 0.03% (v/v) H2O2 and 0.15 mg/ml 4-chloro-1-naphthol.

Flow cytometric analysis and fluorescence-activated cell sorting

For flow cytometric analysis, cultures of BMH71-18 containing the respective expression plasmid were grown overnight at 37°C and subcultured 1:50 at 37°C until they reached an OD600 of 0.2. After induction with anhydrotetracycline (0.2 µg/ml) for 30 min, cells (1 ml) were pelleted by centrifugation in a table-top centrifuge for 1 min and resuspended in 10 µl of PBSS (PBS containing 0.2 M sucrose). After addition of 1 µl of the respective antibody (1 mg/ml), cells were incubated for 5 min at room temperature. After addition of 300 µl of PBSS, cells were centrifuged for 1 min and resuspended in 10 µl of anti-mouse IgG (whole molecule)–biotin conjugate (Sigma B-7264), diluted 1:10 in PBSS. After 5 min of incubation at room temperature and addition of 300 µl of PBSS, cells were pelleted again and resuspended in 5 µl of PBSS containing streptavidin–R-phycoerythrin conjugate (Molecular Probes) at 100 µg/ml, followed by 5 min of incubation at room temperature. Finally, after addition of 300 µl of PBSS, cells were pelleted and resuspended in 1 ml of PBS for flow cytometric analysis. At least 500 000 events were collected on a MoFlo cell sorter (Cytomation). Parameters were set as follows: forward scatter, side scatter, 730 (LIN mode, amplification factor 6); FL1 (FITC), 600 (LOG mode); FL2 (PE), 600 (LOG mode); and trigger parameter, side scatter. The sample flow rate was adjusted to an event rate of ~30 000 s–1.

For fluorescence-activated cell sorting, cells of BMH71-18 harboring pASK21T-EETI–CKSend or pASK21T-EETI–CKEtag, respectively, were grown overnight at 37°C and subcultured 1:50 at 37°C until they reached an OD600 of 0.2. After induction with anhydrotetracycline (0.2 µg/ml) for 40 min, both strains were mixed at a 107:1 ratio and cells from 50 ml of the mixed bacterial liquid culture were pelleted by centrifugation at 4000 g for 10 min. After washing with 1 ml of PBSS, cells were incubated with 100 µl of anti-E antibody (100 µg/ml) for 5 min. Cells were pelleted by centrifugation, washed with 1 ml of PBSS and incubated with 100 µl of anti-mouse IgG (whole molecule)–biotin conjugate (Sigma B-7264), diluted 1:10 in PBSS. After pelleting and washing, cells were resuspended in 100 µl of PBSS containing 20 µl of colloidal streptavidin superparamagnetic microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). After 2 min of incubation at 4°C, cells were pelleted, washed with 1 ml of PBSS and resuspended in 100 µl of PBSS containing streptavidin–R-phycoerythrin conjugate (Molecular Probes) at 100µg/ml, followed by 5 min of incubation at room temperature. Finally, cells were pelleted, washed with 1 ml of PBSS and resuspended in 0.5 ml of PBSS. A MiniMACS high gradient magnetic separation column (Miltenyi Biotech) was attached to a MiniMACS magnet (Miltenyi Biotech) and washed with 4x500 µl of PBSS before separation. The labeled cell suspension was applied to the column and the flow-through was collected and re-applied. After rinsing the column with 4x500 µl of PBSS, the column was removed from the magnet. Bound cells were flushed out by applying 2x1 ml of PBSS to the column with gentle pressure using the plunger supplied with the column. For determination of the number of living cells before and after separation, serial dilutions of a small aliquot of the respective bacterial cell suspensions were plated on to chloramphenicol-containing agar plates.

The bacterial cells eluted from the MiniMACS column were subjected to fluorescence-activated cell sorting using a sorting gate ranging from 19.5 to 1000 relative fluorescence units (compare Figure 7Go). By varying the sample pressure of the Cytomation MoFlo cell sorter, the event rate was adjusted to ~30 000 s–1. Cells were sorted in `enrich 3' mode, immediately resorted, transferred into 50 ml of dYT containing chloramphenicol at 25 µg/ml and grown overnight at 37°C. After induction, 1 ml of the cell culture was labeled as described above and 2x107 cells were sorted in `enrich 3' mode, immediately resorted, transferred into 50 ml of dYT containing chloramphenicol at 25 µg/ml and cultivated overnight at 37°C.


    Acknowledgments
 
We thank A.Skerra for the gift of plasmid vector pASK75, G.Georgiou for the gift of pTX21 and W.Kramer for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through grant Ko 1390/5-1.


    Notes
 
2 Present address: Institut für Anorganische Chemie, Georg-August-Universität, Tammannstrasse 2, D-37077 Göttingen, Germany Back

3 To whom correspondence should be addressed. E-mail: hkolmar{at}uni-molgen.gwdg.de Back


    References
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 Results
 Discussion
 Materials and methods
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Received February 10, 1999; revised June 4, 1999; accepted June 11, 1999.