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

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 beta-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 beta-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 10(7)-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.     

Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.

We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies.     

New binding specificities derived from Min-23, a small cystine-stabilized peptidic scaffold

The randomization of both internal and surface residues in small protein domains followed by selection from a display library is emerging as a powerful strategy to obtain novel binding specificities. Small and stable scaffold motifs observed in disulfide-rich proteins are attractive because they are small, stable, and accessible to chemical synthesis. The elementary structural motif found in the squash trypsin inhibitor EETI-II (Ecballium elaterium trypsin inhibitor) is the cystine stabilized beta-sheet (CSB) motif, found in nearly 50% of all known small disulfide-rich protein families. We have used Min-23, a short 23-residue peptide containing the CSB motif and shown to be a stable autonomous folding unit and one of the smallest scaffolds described to date, as a scaffold for selection of new binding ligands. We demonstrate that the core CSB motif in Min-23 is permissive to loop insertion, using peptide epitopes from hemagglutinin (HA) and Gla-protein (E). A phage library of more than 10(8) different clones has been constructed by insertion of a randomized sequence on a beta-turn of the Min-23 peptide. The selection of this library on a variety of 7 different targets allowed the isolation of 21 new specific binders, confirming the potential of Min-23 as a scaffold for the development of new ligands. The derived library is able to provide a wide range of novel compounds with possible applications in various biological and pharmaceutical areas.     

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒（HTNV）核衣壳蛋白（NP）B细胞抗原表位。以抗HTNV NP单克隆抗体(mAb）5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库。阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV76－118株S基因进行同源性分析。结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制。10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76－118株NP氨基端的aa25-33一致。证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定。     

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.     

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.ABBREVIATIONS USED: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay; PBS, phospho-buffered saline; cfu, colony forming units.     

Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope

Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.     

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix–loop–helix peptides

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix–loop–helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein

Background

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Methodology/Principal Findings

Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α_vβ₃ and α_vβ₅ integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α₅β₁ and α_iibβ₃. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α_vβ₃ and α_vβ₅ integrins. A ⁶⁴Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.

Conclusions/Significance

We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.     

Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.     

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗2B4对噬菌体随机12肽库进行亲和筛选,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆.经三轮筛选后,与抗体结合的噬菌体得到明显富集,噬菌体ELISA结果显示,阳性率达80%.将其中12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验,抑制作用非常明显,有良好的剂量依赖关系,证明这12个克隆与LPS具相似表位.DNA测序并推导噬菌体展示肽的氨基酸序列为,GPPQWFFSQPQL（5/12,41.7%）,LPQYFWNTATTA（3/12,25%）,FPQNHWNVPWAT（2/12,16.6%）,HSQSFWNAPLAM和AHPWTHGYFPPL（1/12,8.3%）.实验结果表明,用2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位,即脂多糖的模拟肽（位）.     

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.     

Different immune response of mice immunized with conjugates containing multiple copies of either consensus or mixotope versions of the V3 loop peptide from human immunodeficiency virus type 1

The critical role that antibody responses to the V3 loop epitope play in human immunodeficiency virus type 1 (HIV-1) neutralization has caused this peptide to be used in many HIV-1 vaccine candidates. To enhance cross-reactivity toward several V3 sequences, a database of 50 peptides of the V3 region from HIV-1 subtype A was used to design both a consensus peptide and a combinatorial peptide (mixotope) library representative of these sequences. The two immunogens (consensus and mixotope) were incorporated into multiple antigen peptide (MAP) constructions, conjugated to a recombinant surface antigen from hepatitis B virus (HbsAg) carrier protein, and inoculated to mice in combination with a C4 (CD4-binding) peptide MAP construction, also conjugated to HBsAg. The respective responses and cross-reactivity to several V3 loop sequences of both types of immunogens were compared. Mice inoculated with the V3 consensus-MAP-HBsAg + C4-MAP-HBsAg mixture elicited higher antibody responses than those given the V3 mixotope-MAP-HBsAg + C4-MAP-HBsAg mixture. In addition, pooled serum from the first group of immunogens analyzed at dilution 1:100 had higher cross-reactivity against V3 peptides on cellulose membranes than those from mice given the combinatorial immunogen. Fine epitope mapping of both consensus and C4 peptide by the spot synthesis technique showed that sera of the first group strongly recognized both sequences in their entirety, whereas mice immunized with the mixotope library recognized only the N-terminal region of V3. These results seem to suggest that the V3 consensus peptide is superior to the combinatorial strategy in inducing potent and cross-reactive responses to HIV.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.     

Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.     

Investigation and molecular mimicry of the antigen involved in the interaction between the monoclonal antibody 5D10 and the human breast cancer cell line MCF-7

Monoclonal antibody (mAb) 5D10 is directed against the human breast cancer cell line MCF-7. Biochemical characterization of the antibody epitope was attempted and revealed a complex, most likely carbohydrate-linked nature, which prevented isolation and further studies of the interaction. A major goal of this work was to generate structural mimics of the 5D10 epitope to serve as putative substitutes in such studies. A peptide library displayed on filamentous phage was used to select for mimotope peptide sequences. All positive phage clones selected from the library displayed the amino acid sequence H(2)N-QMNPMYYR-CO(2)H. This peptide sequence, as well as a branched form of the peptide, was found to bind mAb 5D10. Moreover, both peptide sequences were able to inhibit the binding of 5D10 to the MCF-7 cells in a concentration-dependent manner, with an EC(50) value in the range of 65 microM. According to these results, random phage peptide libraries can serve to identify mimotopic peptides for unknown complex cell surface epitopes.     

利用15肽随机肽库确定抗TNF单抗表位的研究

利用抗ＴＮＦ的Ｔ５单抗作为筛选配基,对经ＤＮＡ碱基组成分析证明具有良好随机性的１５肽库进行亲和筛选．经过三轮筛选后,以硝酸纤维素膜斑点印迹法观察到良好的富集效果．由第三轮挑选出的３１个克隆进行ＤＮＡ测序,结果推出的优势克隆的短肽为ＣＹＲＲＰＡＧＧＬＰＧＩＣＳＡ等,竞争性ＥＬＩＳＡ实验证明带有以上短肽的噬菌体与ＴＮＦ能竞争性地与Ｔ５单抗结合．该多肽可能是Ｔ５单抗所识别的模拟表位     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

Cyclic peptides selected by phage display mimic the natural epitope recognized by a monoclonal anti-colicin A antibody.

A 10-mer random peptide library displayed on filamentous bacteriophage was used to determine the molecular basis of the interaction between the monoclonal anti-colicin A antibody 1C11 and its cognate epitope. Previous studies established that the putative epitope recognized by 1C11 antibody is composed of amino acid residues 19-25 (RGSGPEP) of colicin A. Using the phage display technique it was confirmed that the epitope of 1C11 antibody was indeed restricted to residues 19-25 and the consensus motif RXXXPEP was identified. Shorter consensus sequences (RXXPEP, RXXEP, KXXEP) were also selected. It was also demonstrated that the disulfide bond found in one group of the selected peptides was crucial for 1C11 antibody recognition. It was shown that cyclization of the peptides by disulfide bond formation could result in a structure that mimics the natural epitope of colicin A.