Minor Coat Protein Research Articles

Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library.

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

Antibodies from phage display libraries as immunochemical reagents.

AbstractThe display of a repertoire of recombinant antibodies on the surface of filamentous phage by fusion to a minor coat protein has provided a powerful methodology for isolating the desired binding specificities (see Chapter 45 and ref. 1). Antibodies have typically been displayed on phage as single-chain Fv fragments (scFv [2]), in which the heavy chain variable region (VH) and the light chain variable region (VL) domains are linked together by a flexible polypeptide (3,4), or as Fab fragments (5) In general, antibodies have been displayed either at the N-terminus of the minor coat protein pill of filamentous phage (2), or at the C-terminal domain of pill (6). For Fab fragments, one chain is fused to pill, and the other assembles with the pill-fused chain after leader peptide-mediated secretion in the bacterial supernatant.KeywordsAntibody FragmentPhage ParticleFilamentous PhageHelper PhagePhage ELISAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Emerging Impact of Phage Display Technology in Thrombosis and Haemostasis

IntroducationThe phage display technology represents a powerful tool for protein and drug design because vast numbers of amino acid sequences can be rapidly explored. This review describes the origins of phage libraries, their evolution and more recent advances, including examples in the area of thrombosis and haemostasis, where the phage display approach has just begun to be used with great success.Phage display uses filamentous phages (E. coli specific phages with a filamentous shape that contain a single stranded closed circular molecule of DNA), such as M13 or fd, as vehicles for displaying foreign peptides or proteins on their surface. This is carried out by fusing the coding sequence (DNA) for the peptide or protein to the amino (N)-terminus of either full-length phage minor coat protein III (cpIII), or to phage major coat protein VIII (cpVIII), to carboxy (C)-terminal domain of cpIII or, more recently, by fusion to the C-terminus of full-length phage minor coat protein VI (cpVI) (Fig. 1). Expression of the fusion protein and its subsequent incorporation into the mature phage particle results in the foreign peptide or protein being presented on the phage surface. Thus, the linkage of each peptide or protein to its encoding genetic material [contained as part of the single-stranded viral DNA (1, 2)] represents a great advantage over conventional cloning methods.The phage display approach was first used by Smith in 1985 (3), who expressed a library of peptide sequences at the N-terminus of cpIII (3-5). This insertion allowed phage assembly and display of the peptide on the phage surface, without affecting the phage infectivity significantly. The linkage of genotype and phenotype in the phage library allowed facile isolation of clones of specific interest from pools of millions of clones by successive rounds of phage affinity selection on surfaces coated with a ligand (panning) followed by phage amplification by infecting male E. coli (Fig. 2).The foreign peptides or proteins were displayed, initially, on every copy of the coat protein, but only short peptides can be displayed in this way without altering the phage infectivity. Two systems have been developed to solve this problem. One incorporates a second native gene III or VIII in the phage genome giving a mixture of native and recombinant coat protein incorporation on the phage. The second system provides the recombinant cpIII, cpVIII or cpVI gene on a phagemid (a plasmid containing the origin of replication of filamentous phage). Phagemids can be packaged into phage particles by superinfection with a helper phage. The fusion protein is incorporated onto the surface coat, along with copies of the native coat protein encoded by the helper phage. The result is a mixture of wild-type helper phage and recombinant phagemid particles, but due to a defective origin of replication the helper phage is poorly packaged to provide minimal competition with the phagemids (6). Phages that display both native coat protein and fusion protein are infective. Therefore, the display systems can be either multivalent or monovalent. Multivalent systems make use of gene III phage constructs or gene VIII phage or phagemid constructs and give a high number of foreign domains displayed on their surface (7). Monovalent systems utilize gene III or gene VI phagemid constructs, which have a low number of foreign domains displayed on their surface, usually a single copy. Consequently, monovalent systems distinguish between low affinity and high affinity clones in panning assays (1, 8).

Identification of Gene VI of Filamentous Phage φLf Coding for a 10-kDa Minor Coat Protein

ORF95 in the filamentous phage φLf genome, locating behindgIII,was identified to be the gene (gVI) coding for minor coat protein pVI (95 amino acids, 10,245 dal). It was shown to be virion associated by Western blot analysis of chloroform-treated phage particles. Computer analysis predicted two transmembrane regions for this protein. Since no signal peptide was suggested and the size estimated by SDS-polyacrylamide gel electrophoresis matches that deduced from nucleotide sequence, it appears to be incorporated into the phage particle as its primary translational product. After completion of this study, eight genes organizing into an order ofgII-gX-gV-gVII-gIX-gVIII-gIII-gVIhave been identified for φLf.

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (V H) and light (V L) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

The C-Terminal Domain of TolA Is the Coreceptor for Filamentous Phage Infection of E. coli

Filamentous bacteriophages infecting gram-negative bacteria display tropism for a variety of pilus structures. However, the obligatory coreceptor of phage infection, postulated from genetic studies, has remained elusive. Here we identify the C-terminal domain of the periplasmic protein TolA as the coreceptor for infection of Escherichia coli by phage fd and the N-terminal domain of the phage minor coat protein g3p as its cognate ligand. The neighboring g3p domain binds the primary receptor of phage infection, the F pilus, and blocks TolA binding in its absence. Contact with the pilus releases this blockage during infection. Our findings support a sequential two-way docking mechanism for phage infection, analogous to infection pathways proposed for a range of eukaryotic viruses including herpes simplex, adenoviruses, and also lentiviruses like HIV-1.

A Selection System to Study Protein–RNA Interactions: Functional Display of HIV-1 Tat Protein on Filamentous Bacteriophage M13

The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle and belongs to the RNA binding proteins of the arginine-rich motif (ARM) family. Very little is known about their mechanism of RNA recognition. To study the principles of RNA-protein recognition we constructed a system to display HIV-1 Tat on the surface of the filamentous bacteriophage M13. HIV-1 Tat (1-72) and a mutant Tat lacking five cysteine residues were cloned into the pAK phagemid system, which allows fusion of thetatgene to a supershort version of the gene for minor M13 coat protein. Expression of the resulting fusion proteins was shown via western blot analysis. Phages displaying functional Tat could be selected from phages without Tat or with a non-functional Tat variant via binding to biotinylated TAR using streptavidin coated paramagnetic beads. By randomizing certain amino acid positions of Tat and screening of the resulting phage libraries for affinity and specificity, we are now able to study the role and importance of amino acids of HIV-1 Tat for affinity and specificity to TAR RNA.

Phage-displayed La/SS-B antigen as a diagnostic reagent

Background: The detection of antibodies to La/SS-B, a nuclear RNA-binding protein in mammalian cells, aids in the diagnosis of Sjogren's syndrome and systemic lupus erythematosus (SLE). This is performed conventionally by immunoprecipitation using a crude splenic extract and more recently, by the more sensitive and rapid enzyme-linked immunosorbent assay (ELISA) which uses a purified La/SS-B antigen. The latter antigen is obtained from cellular extracts of the antigen or from bacterial cell lysates containing the recombinant antigen usually by affinity chromatographic method. Objective: To produce a La/SS-B antigen for use in ELISA that can be obtained easily and inexpensively without the need for extensive purification (including affinity chromatography). Study design: The antigen was produced as a fusion protein of the minor coat protein of M13 bacteriophage and used in this phage-associated form in an ELISA. La/SS-B cDNA derived from Hep-2 cells was cloned into the phagemid, pCANTAB-5E, and transfected to Escherichia coli. Phage clones selected for the presence of insert both by gene and antigenic analyses were used in the ELISA to detect anti-La/SS-B antibodies from patients with Sjogren's syndrome and SLE. Results: A phage clone was obtained which contained a La/SS-B cDNA fragment truncated at the C-terminal end (after base-pair 631). The phage-displayed antigen derived from this clone was obtained by precipitation of the phage particles from the bacterial culture supernatant with polyethylene glycoI. Used in the ELISA, this antigen detected 27 of 28 precipitin-positive sera and was negative for 50 control sera. The soluble (phage-free) form of the antigen was obtained from a nonsuppressor host as a cell lysate which could not be used in this form in an ELISA for antibody detection. It was useable, however, in Western blot analysis which confirmed the reactivity of the recombinant antigen. Conclusion: Phage-displayed antigens may be used in place of soluble forms of these antigens in detection assays which have the advantage that they are easy and inexpensive to produce.

Identification of a surrogate TSL-1 antigen of Trichinella spiralis from a phage library of random peptides.

We sought to find a peptide analogue of an important antigen (TSL-1) of Trichinella spiralis which is recognized by the 7C2C5 antibody. A phage library which displays a short (15-mer) randomly-generated peptide at the filament of the minor coat protein of the virion was used for selection by the 7C2C5 antibody. A peptide thus identified, ICDASGLGCWCWSLSP, was found to be a true surrogate since its binding to the antibody could be blocked by the native antigen and, conversely, an antiserum made to the peptide could recognize the native antigen. In addition, the peptide appeared to detect T. spiralis-infected pigs although it was less discriminatory than the native antigen.

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of CAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by displaying it on the surface of filamentous bacteriophage fd. Phosphorylationin vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-rner peptide library phosphorylated by cAPK was selected with ferric (Fe(3+)) chelation affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Theirin vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe(3+)) chelation affinity chromamraphy to identify the substrate specificity of protein kinase from random peptide library was feasible.

The Functional Display of Interleukin-2 on Filamentous Phage

We report the novel display of interleukin-2 (IL-2) and an IL-2 analog, D126, on the surface of filamentous bacteriophage using a phagemid vector system. A synthetic human IL-2 gene and its D126 analog were fused to the carboxyl-terminal domain of the gene III minor phage coat protein. Expression of IL-2 and D126 was verified by their reactivity with an IL-2-specific antibody. Biological response of IL-2 phage on murine CTLL-2 cells was comparable to that of recombinant soluble IL-2, while the D126 phage displayed a reduced biological response similar to that previously measured by soluble D126 protein. Biosensor surface plasmon resonance was employed to verify binding of the IL-2 and D126 phage to the IL-2 αβcc receptor complex. A 41-fold enrichment of IL-2 phage over R408 helper phage was demonstrated in biopanning affinity selection studies employing biotinylated αβcc receptor complex. These biopanning studies are the first reports of affinity selection of IL-2 phage and demonstrate a novel use for the αβcc receptor complex. Together, these studies confirm that the structural integrity of IL-2 and D126 is maintained when they are displayed as a gIIIp fusion protein on phage particles and provide the foundation for further selection studies employing IL-2 analog phage libraries.

Enzymatic properties of phage-displayed fragments of human plasminogen.

Two low-molecular-mass forms of human plasminogen, plasminogen-(543-791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444-791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen-pIII/phage particle and 0.1 molecules mini-plasminogen-pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human alpha2-antiplasmin or the inhibitor Phe-Pro-Arg-CH2Cl. Micro-plasminogen-pIII was activated by streptokinase and staphylokinase, two non-enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini-plasminogen-pIII micro-plasminogen-pIII and mini-plasminogen dissolved 125I-labelled fibrin films in a dose-dependent time-dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The micro-plasminogen and mini-plasminogen phage-display systems may be useful to study mechanisms of plasminogen activation.

Comparison of promoters and selectable marker genes for use in Indica rice transformation

Key words: bar, embryogenic callus, hph, Indica riceAbstractThe effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encodingthe Escherichia coli uidA(gus) gene under the control of CaMV 35S, Emu,Act1 or Ubi1 promoters were deliveredinto cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatestfrom plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissuewas examined by bombarding embryogenic Indica rice calli with a pUbi1-gusplasmid and a plasmid containingeitherthe selectable markergene,hph, whichconfershygromycinresistance, orbar, whichconfersresistance to theherbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters.The bombardedcalli were placed on the appropriateselection media and stained for GUS activity at 1 day, 3 weeksand 5 weeks after shooting. Callus bombarded with the pUbi1-hphor the pEmu-hph constructs gave a dramaticincrease in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas wasobserved in calli co-bombarded with pUbi1-gusand any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hphconstruct and a virus minor coat protein (cp)gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hphand cp genes. These genes were stably inherited by the T

Phage-displayed Fab fragments against anti-human interleukin-2 receptor alpha. Detection of antigen-bound phages with anti-cpIII monoclonal antibodies.

The genes encoding the VHCH1 and VLCL parts of the mouse anti-human IL-2R alpha antibody 7G7B6 were amplified by PCR and the corresponding antibody fragments displayed on the surface of filamentous phages. The expression of Fab fragments was analysed by immunoblotting using HRP-labelled goat anti-mouse Ig antisera. By traditional hybridoma technology, splenocytes from Balb/c mice, immunized with native phage particles, were fused with P3X63-Ag8.653 myeloma cells in order to yield monoclonal antibodies against filamentous phage proteins. The obtained monoclonal antibody IF8 (mu/kappa) recognized the minor coat protein III as a 65-70 kDa protein band by immunoblotting, whereas the monoclonal antibody IVC8 (mu/kappa), in addition to cpIII, recognized a protein with an approximate molecular weight of 38-43 kDa. Both antibodies were employed to determine the binding specificity of the phage-displayed anti-human IL-2R alpha Fab fragments in an ELISA using recombinant baculovirus-expressed human IL-2R alpha proteins as antigens.

Bacteriophage Display of Chymotrypsin Inhibitor 2

Abstract: A chymotrypsin inhibitor 2 variant, CI2(G83D), was expressed as a fusion with fd minor coat protein, pili, via a flexible linker (Gly-Ala 3) and displayed on the surface of bacteriophage. Biopanning with immobilised subtilisin BPN' demonstrated that the inhibitor-displaying phage could be selectively enriched 13-fold over control phage in one round of affinity purification. The inhibitor (incorporating the linker sequence) was also expressed as soluble protein in E. coli and purified to homogeneity. Kinetic analysis revealed the displayed inhibitor possesses inhibition characteristics that approximate those of this soluble inhibitor. The results demonstrate that functionally active inhibitor can be displayed on the phage surface. Further use of this technology should facilitate the selection of remodelled inhibitor variants exhibiting novel protease binding specificities.

Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis

Background: The anaphylatoxin C5a is a powerful proinflammatory protein generated on activation of the complement system. Recently, we described an anti-hC5a neoepitope specific mAb, mAb 2925, which was raised against the nonapeptide ISHKDMQLG (C5a-(65–73). This mAb is unique in that it recognizes both hC5a and hC5adesArg, even when it is denatured. It inhibits binding of [125I]C5a to its receptor on Bt2-CAMP differentiated U937 cells. Objectives: To define the epitope of mAb 2925, we used a combined approach of a bacteriophage random octapeptide library, synthetic peptides and site-directed mutagenesis. Study design: First a phage peptide library was screened with the anti C5a mAb 2925. Then synthetic peptides were synthesized with respect to the sequence information yielded from the phage approach, and used for binding studies. Site-directed mutagenesis was performed to confirm the results from the mapping experiments. Results and conclusion: Most phages selected by biotinylated Fab 2925 displayed sequences on the minor coat protein which correspond to residues within the C-terminus of human C5a. A first consensus motif comprised amino acids His-Lys or His-Arg, which allowed us to define position 67 and 68 as part of the epitope. A second consensus motif was selected, comprising Arg/Lys-Trp-Tip. This motif did not match any residues within the C5a C-terminus. However, when expressed together with the consensus motif His-Arg, as in HRWWXXXX or in HRXKWWXX, binding of these peptides to Fab 2925 increased as compared to peptides expressing the His-Arg motif only. Thus, the Arg/Lys-Trp-Tip motif serves to stabilize the binding of His-Arg to mAb 2925. Synthetic peptide studies revealed further N-terminal residues Ile65 and Ser66 as part of the epitope. A C5a mutant with an exchange Lys68Glu (C5aGlu68) confirmed the participation of Lys68 as a contact residue within the epitope of mAb 2925. Hence, the epitope recognized by mAb 2925 is linear and comprises residues Ile65, Ser66, His67, and Lys68. Thus, we could demonstrate for the first time that a mAb inhibits C5a receptor binding through specific interaction with receptor binding residues of the ligand.

PJuFo: a phagemid for display of cDNA libraries on phage surface suitable for selective isolation of clones expressing allergens.

We have developed a phagemid vector (pJuFo) for the selective isolation of genes encoding proteins for which a ligand is available. The physical linkage of cDNA-encoded proteins to the genetic information required for their production was achieved by exploiting the high affinity interaction of the Jun and Fos leucine zippers. The gene encoding Jun preceeded by the pelB leader sequence placed under control of a lac promoter/operator element was cloned as N-terminal fusion to the minor viral coat protein gIII. The Jun/gIII fusion protein become structurally incorporated into phage particles during infection with helper phage. The gene encoding Fos, proceeded by the pelB leader sequence was coexpressed from a second lac promoter/operator as N-terminal fusion peptide to cDNA encoded proteins. The resulting Fos-fusions secreted into the periplasmic space of E. coli could become associated with Jun-decorated phage particles and thus displayed on phage surface. To avoid dissociation of the Jun/Fos heterodimeres cysteines were engineered at the N- and C-termini of both leucine zippers providing a covalent link of the cDNA gene products to the genetic information required for their production. The vector allows the construction of phage display cDNA expression libraries which can be screened by biopanning in microtiter plates coated with a ligand exhibiting affinity to the gene products of interest. The expression products from a cDNA library from Aspergillus fumigatus, a mold known to produce more than 40 IgE binding proteins, have been displayed on the surface of phage Ml3 using pJuFo. Phage displaying IgE-binding proteins were selectively enriched over non-specific phage by consecutive rounds of growth and selection in a microtiter plate coated with serum IgE from allergic individuals. A vast variety of phage encoding and displaying different IgE-binding proteins with an apparent molecular mass ranging from 20 to > 40 kDa were isolated from this library.KeywordscDNA LibraryPhage DisplayPhage ParticlePhage Display LibrarySelective IsolationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

P-C4-31 Properties of the membrane-bound forms of the gene VII and gene IX minor coat proteins of bacteriophage M13: Houbiers MC, Spruijt RB, Wolfs CJAM, Hemminga MA Department of Molecular Physics, Agricultural University, Wageningen, (NL)

P-C4-31 Properties of the membrane-bound forms of the gene VII and gene IX minor coat proteins of bacteriophage M13: Houbiers MC, Spruijt RB, Wolfs CJAM, Hemminga MA Department of Molecular Physics, Agricultural University, Wageningen, (NL)

Reiterative screening of phage-display peptide libraries with antibodies.

Filamentous bacteriophage can tolerate the insertion of a foreign DNA into their gene encoding phage minor coat protein III. Such recombinant phage particles display on their surface foreign peptide sequences fused with the gene III product, which is a protein represented by five copies on the top of the virion. The N-terminal part of this coat protein (cp III) is involved in binding phage to the F-pilus of a bacterium at an early stage of the infection process. The exposed peptide region exists, therefore, in a form accessible for immune recognition (). Modern gene engineering allows the creation of phage libraries in which every possible combination of amino acid sequence is expressed as fusion proteins (, , , ). An important difference between random peptides and specific peptides (e.g., PEPSCAN) or antigen fragmentation methods is that the former not only localizes the epitope in the antigen sequence, but also provides information on which amino acid residues are most important for antibody binding.