A general route to fingerprint analyses of peptide-antibody interactions using a clustered amino acid peptide library: Comparison with a phage display library

1995 ◽  
Vol 32 (7) ◽  
pp. 459-465 ◽  
Author(s):  
Achim Kramer ◽  
Evangelia Vakalopoulou ◽  
Wolf-Dieter Schleuning ◽  
Jens Schneider-Mergener
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Phage Display Library ◽  
Peptide Library ◽  
Amino Acid Peptide ◽  
Peptide Antibody ◽  
Antibody Interactions

scholarly journals Citrullination of a phage displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies

2021 ◽  
Author(s):  
Gabriel D. Román-Meléndez ◽  
Daniel R. Monaco ◽  
Janelle M. Montagne ◽  
Rachel S. Quizon ◽  
Maximilian F. Konig ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Amino Acid ◽  
Phage Display ◽  
Phage Display Library ◽  
Human Proteome ◽  
Phage Display Technology ◽  
Post Translational Modifications ◽  
Amino Acid Peptide ◽  
Display Technology ◽  
Citrullinated Protein

AbstractPost-translational modifications (PTMs) on proteins can be targeted by antibodies associated with autoimmunity. Despite a growing appreciation for their intrinsic role in disease, there is a lack of highly multiplexed serological assays to characterize the fine specificities of PTM-directed autoantibodies. In this study, we used the programmable phage display technology, Phage ImmunoPrecipitation Sequencing (PhIP-Seq), to profile rheumatoid arthritis (RA) associated anti-citrullinated protein antibody (ACPA) reactivities. Using both an unmodified and peptidylarginine deiminases (PAD)-modified phage display library consisting of ~250,000 overlapping 90 amino acid peptide tiles spanning the human proteome, PTM PhIP-Seq robustly identifies antibodies to citrulline-dependent epitopes. PTM PhIP-Seq was used to quantify key differences among RA patients, including PAD isoform specific ACPA profiles, and thus represents a powerful tool for proteome-scale antibody-binding analyses.

scholarly journals Tight-binding inhibitory sequences against pp60c−srcidentified using a random 15-amino-acid peptide library

FEBS Letters ◽  
1996 ◽  
Vol 399 (3) ◽  
pp. 237-240 ◽  
Author(s):  
Toru Nishi ◽  
Raymond J.A. Budde ◽  
John S. McMurray ◽  
Nihal U. Obeyesekere ◽  
Naueen Safdar ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tight Binding ◽  
Peptide Library ◽  
Amino Acid Peptide

Amino acid determinants of β-hairpin conformation in erythropoeitin receptor agonist peptides derived from a phage display library

2002 ◽  
Vol 316 (5) ◽  
pp. 1111-1125 ◽  
Author(s):  
Nicholas J Skelton ◽  
Stephen Russell ◽  
Frederic de Sauvage ◽  
Andrea G Cochran
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Receptor Agonist ◽  
Phage Display Library ◽  
Hairpin Conformation

scholarly journals Investigation of antigen-antibody interactions using a soluble, non-support-bound synthetic decapeptide library composed of four trillion (4 × 1012) sequences

1994 ◽  
Vol 301 (3) ◽  
pp. 847-853 ◽  
Author(s):  
C Pinilla ◽  
J R Appel ◽  
R A Houghten
Keyword(s):  
Antigenic Determinant ◽  
Peptide Library ◽  
Antibody Recognition ◽  
C Terminus ◽  
Peptide Antibody ◽  
Positional Scanning ◽  
Ic50 Values ◽  
Antigen Antibody ◽  
Antibody Interactions ◽  
Specific Sequences

A decapeptide positional-scanning synthetic-peptide combinatorial library (PS-SPCL) made up of four trillion (4 x 10(12) decapeptides was synthesized; its use is illustrated here for the study of a peptide-antibody interaction. This library was prepared by a chemical-mixture approach using a specific ratio of amino acids empirically determined to give approximately equimolar incorporation of each amino acid during each coupling step. Despite the immense number of decapeptides making up each peptide mixture [approx. 200 billion (2 x 10(11)], specific sequences having nanomolar affinities for a peptide-antibody interaction could be readily identified. Upon screening this decapeptide PS-SPCL in this well characterized system, the known six-residue antigenic-determinant sequence was found, with the most specific residues appearing to ‘walk through’ the ten positions of the peptide library. More importantly, it appears that antibody recognition in this system is stronger when the antigenic determinant is located at the C-terminus of the decapeptide library. Individual decapeptides corresponding to sequences derived from the most active peptide mixtures at each position were synthesized to confirm the results of the screening; 15 peptides were found to have IC50 values between 0.6 and 9.5 nM, four of which were found to be 5-10 times more active than the known six- and 13-residue control peptides. These results further illustrate the power of the positional-scanning peptide library concept, and extend its practical range to a decamer library composed of four trillion (4 x 10(12) decapeptides.

scholarly journals Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
David N. Hernandez ◽  
Kayan Tam ◽  
Bo Shopsin ◽  
Emily E. Radke ◽  
Pegah Kolahi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Phage Display ◽  
B Cell ◽  
Three Dimensional ◽  
Amino Acid Side Chain ◽  
Gene Fragment ◽  
B Cell Epitopes ◽  
Content Type ◽  
Amino Acid Peptide

ABSTRACT Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

Screening and identification of the mimic epitope of the adhesion protein of Mycoplasma genitalium

2012 ◽  
Vol 58 (7) ◽  
pp. 898-908 ◽  
Author(s):  
Yanhua Zeng ◽  
Liangzhuan Liu ◽  
Jun He ◽  
Yan Liu ◽  
Cuiming Zhu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Mycoplasma Genitalium ◽  
Phage Display Library ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Adhesion Protein ◽  
Phage Display Peptide Library ◽  
Screening And Identification ◽  
Peptide Phage Display

Mycoplasma genitalium adhesion protein (MgPa) is the major adhesion protein of M. genitalium, and its C-terminal domain (amino acid 1075–1444) is the most immunogenic region. However, the exact epitopes of the adhesion protein of M. genitalium are still unclear. We used the purified polyclonal antibody against the recombinant adhesion protein to screen the mimic epitopes of MgPa using a random 12-peptide phage display library. Immunoscreening via the phage display peptide library revealed that 3 motifs (P-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L, and K-S-L-S-R-X-D-X-I) may represent 3 different mimotopes of MgPa. Results of bioinformatics analysis by MIMOX demonstrated that the key consensus amino acid residues in the aligned mimotopes may be S, A, and F for cluster 1; A, K, I, T, and L for cluster 2; and K, S, L, R, D, and I for cluster 3. Three representative phages could recognize sera from M. genitalium-positive patients to varying degrees, whereas they could not recognize the sera from Mycoplasma pneumoniae -positive patients or the sera from healthy people. These findings will help to clarify the mimic epitopes of MgPa to facilitate diagnosis of the antigen and to understand the antigenic structure of MgPa.

scholarly journals Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

2003 ◽  
Vol 50 (3) ◽  
pp. 857-864 ◽  
Author(s):  
Piotr Mucha ◽  
Agnieszka Szyk ◽  
Piotr Rekowski ◽  
Paul F Agris
Keyword(s):  
Capillary Electrophoresis ◽  
Amino Acid ◽  
Gene Expression Regulation ◽  
Phage Display Library ◽  
Significant Shift ◽  
Mobility Shift ◽  
Amino Acid Peptide ◽  
Peptide Concentration ◽  
Mobility Shift Assay ◽  
Hiv 1

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Characterization of a Human Monoclonal Antibody Directed Against the Spacer Domain of ADAMTS13; a Prototype of Antibodies Present in Patients with Acquired Thrombotic Thrombocytopenic Purpura.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1069-1069
Author(s):  
Brenda M. Luken ◽  
Ellen A.M. Turenhout ◽  
Paul H.P. Kaijen ◽  
Johanna A. Kremer Hovinga ◽  
Jan A. Van Mourik ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Phage Display Library ◽  
Proteolytic Cleavage ◽  
Human Antibody ◽  
Dependent Manner ◽  
Single Chain ◽  
Thrombocytopenic Purpura ◽  
Dose Dependent

Abstract Acquired thrombotic thrombocytopenic purpura (TTP) is often accompanied by the presence of (auto-) antibodies directed against the plasma metalloprotease ADAMTS13. The antibodies prevent efficient proteolytic cleavage of unusually large von Willebrand factor multimers by ADAMTS13, which is highly reactive with platelets. This may result in the formation of platelet aggregates that occlude the microvasculature of patients with TTP. In all presently characterized patients with acquired TTP and detectable anti-ADAMTS13 antibodies, antibodies directed to the (cysteine-rich) spacer domain were present. In order to study anti-ADAMTS13 antibodies at the molecular level, we have isolated single-chain Fv antibody fragments (scFv) from an immunoglobulin V-gene phage-display library derived from CD19+ B-cells of a patient with acquired TTP. From this library scFv I-9 was isolated, of which the variable heavy chain segment is homologous to members of the VH1-69 family. Using ELISA and immunoprecipitation analysis we confirmed that antibody I-9 also binds to the spacer domain. We converted the scFv antibody fragment into a full-length human antibody to better study its effect on proteolytic cleavage of VWF and substrate binding. Antibody I-9 was included into a modified FRETS-VWF73 assay and a dose-dependent inhibition of VWF73 proteolysis by ADAMTS13 was observed. In addition, antibody I-9 was able to inhibit the proteolysis of plasma-derived VWF multimers by ADAMTS13. In both assays inhibition was not very efficient; which is most likely due to the low affinity of antibody I-9 for ADAMTS13. Chimeric ADAMTS13 variants in which 5–10 amino acid residues of the ADAMTS13 spacer domain have been exchanged for the corresponding sequence of ADAMTS1 were used to determine the binding site for antibody I-9 in the spacer domain. Our findings show that amino acid regions 572–579 and 657–666 of the spacer domain are required for binding of antibody I-9, although several other regions are also involved in the interaction. We have previously shown that these amino acid regions are also crucial for binding of (polyclonal) antibodies present in plasma of patients with acquired TTP. Additionally, competition experiments were performed to address whether antibodies with similar properties as antibody I-9 are indeed present in plasma from patients with acquired TTP during acute disease. Plasma from 6 patients with acquired TTP was able to compete in a dose-dependent manner with antibody I-9 for binding to immobilized ADAMTS13, including plasma from the patient whose lymphocytes were used for construction of the phage-display library. These results show that antibodies with similar epitope-specificity as antibody I-9 are present in plasma of patients with acquired TTP. Since anti-spacer domain antibodies have been found in all presently characterized patients with anti-ADAMTS13 antibodies, the current data suggest that antibody I-9 is representative of the pathogenic antibodies present in plasma of the majority of patients with acquired TTP.

BCRs of Mutated and Unmutated CLL Bind Peptides with Distinct Sequence Features: Evidence from Phage Display Libraries.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1118-1118
Author(s):  
Manuela Woelfle ◽  
Till Seiler ◽  
Rosa Catera ◽  
Hartmut Dohner ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Specific Binding ◽  
Phage Display Library ◽  
Amino Acid Sequences ◽  
Phage Display Technology ◽  
Ligand Interactions ◽  
Distinct Sequence ◽  
Peptide Phage Display

Abstract In CLL, the use of specific IgV genes to code the clone’s BCR is non-random and there is an apparent selection for particular genetic and amino acid structures that can be shared by different patients, supporting the hypothesis that antigenic stimulation influences the development and course of CLL. As the binding specificities of the BCR are largely unknown, a vast variety of antigens may affect the BCRs and defined antigens have yet to be identified. Therefore, we used peptide phage display technology to identify ligands for CLL BCRs. BCRs from 2 IgVH unmutated (U-CLL) and 3 mutated (M-CLL) patients were expressed as IgG1 mAbs and used to probe a 12-mer peptide phage display library. In each case, after 3 rounds of selection, we isolated ligands reactive with the CLL mAbs. For the 3 M-CLL mAbs, phage clones carrying peptide inserts with conserved consensus motifs were found. Specificity of the BCR-ligand interactions was demonstrated in direct and indirect ELISA, since selected phage clones and synthetic peptides bound to their respective M-CLL mAb but not to other M-CLL mAbs. Variation of the amino acid sequence of the synthetic peptides significantly altered their reactivity with the corresponding M-CLL mAb. Furthermore, synthetic peptides were bound only by the proper mAb/BCR, but not by mAbs of other M-CLL or U-CLL patients with BCRs comprised of different IgVH genes, supporting the hypothesis that BCRs of M-CLL recognize a defined epitope. In contrast, the mAbs from 2 U-CLL cases did not select phages bearing a consensus motif. Rather these U-CLL mAbs bound multiple phages expressing the same 12-mer peptides, although these differed in sequence between the two U-CLL cases tested. Furthermore, 2 separate selection procedures using 1 U-CLL mAb isolated multiple phage bearing the same 12-mer sequence on each occasion as well as another set of phages with a completely distinct sequence in 1 of 2 selections. ELISAs demonstrated specific binding of all phage clones and of the synthetic peptides by the U-CLL mAbs. Despite this level of specificity, the 2 U-CLL mAbs also reacted with peptides isolated from panning with other CLL mAbs, thereby displaying considerable polyreactivity. Rather than binding only one distinct epitope, mAbs from U-CLL appear capable of interacting with multiple, unrelated structures. Finally, one of the peptides isolated with an U-CLL mAb was bound by all of the CLL mAbs tested, including those from M-CLL cases; therefore this target is antigenically “polyreactive”. Thus, phage display is a feasible approach to identify specific ligands for CLL BCRs. The two classes of BCRs in M-CLL and U-CLL show substantially different binding properties - the former binding shared amino acid motifs and the latter binding multiple ligands of distinct and identical 12-mer amino acid sequences. These peptides can be used to analyze more precisely the binding sites of CLL BCRs as well as the consequences that ensue after BCR crosslinking, and they might help develop BCR-specific therapeutic agents.

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