Combinatorial epitope search: Pitfalls of library design

Yu, Zhiguang; Chu, Yen‐Ho

doi:10.1016/s0960-894x(96)00585-9

Cited by 14 publications

(18 citation statements)

References 7 publications

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“…Synthesis was typically carried out on a 1.0-g scale using standard Fmoc/ HBTU/HOBt chemistry (19) on a homemade peptide synthesis apparatus. For the peptide libraries, a common four amino acid linker, BBRM (B ) β-alanine), was first synthesized on the resin (20). The randomized positions were generated using a split-pool synthesis method to construct the one-bead-one-sequence peptide libraries (21,22).…”

Section: Methodsmentioning

confidence: 99%

Determination of Substrate Specificity for Peptide Deformylase through the Screening of a Combinatorial Peptide Library

Wei

Zhou

et al. 1998

Biochemistry

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Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable of deformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences. In this work, we have developed a combinatorial method to systematically examine the sequence specificity of peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E. coli enzyme. The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides. These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates. In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity. The deformylase was also shown to exhibit esterase activity. These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents. This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases.

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Section: Methodsmentioning

confidence: 99%

Determination of Substrate Specificity for Peptide Deformylase through the Screening of a Combinatorial Peptide Library

Wei

Zhou

et al. 1998

Biochemistry

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“…This library contains all of the possible hexapeptides and each bead carries $100 pmol of a unique peptide sequence. A peptide linker, IBBRM (B b-alanine), was synthesized to the C terminus of the random region (Yu & Chu, 1997), whereas glutamate and aspartate were added to the N terminus of the random region to improve aqueous solubility. Therefore, each member of the library bears the sequence: acetyl-EDXX(pY)XXXIBBRMresin (X random amino acid).…”

Section: Design Synthesis and Screening Of A Ptyr Peptide Librarymentioning

confidence: 99%

“…A common linker of four amino acid residues. BBRM (B b-alanine), was ®rst synthesized on the resin by using a fourfold excess of amino acids (Yu & Chu, 1997). The ®ve random positions were generated by using the split-synthesis method, resulting in a onebead-one-peptide library (Lam et al, 1991;Furka et al, 1991).…”

Section: Synthesis Of the Phosphotyrosyl Peptide Librarymentioning

confidence: 99%

II. Structure and specificity of the interaction between the FHA2 domain of rad53 and phosphotyrosyl peptides

Wang

Byeon

Liao

et al. 2000

Journal of Molecular Biology

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“…Although various dipeptides have been used as spacers in the combinatorial library approach, the glycine-glycine (Gly-Gly) spacer had been recommended as ideal. 38 The advantages of the Gly-Gly spacer is that it projects the epitopic peptide away from the solid support thereby presenting it for antigen-antibody interaction in a specific manner useful for screening by solid phase enzyme-linked immunosorbent assay (ELISA) without affecting the conforma-tion of the epitope. The same spacer was used for the synthesis of peptides of epitopes of infectious bursal disease (IBD), peste des petitis ruminants (PPR) and rinderpest (RP) viruses successfully.…”

Section: Linker or Spacermentioning

confidence: 99%

Diagnostic and immunoprophylactic applications of synthetic peptides in veterinary microbiology

Saravanan

Kumar

2009

Microbiol Res (Pavia)

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Diagnosis of invasive fungal diseases remains problematic, especially in undeveloped countries. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Histoplasma capsulatum using metaperiodate treated purified histoplasmin (ptHMIN). Our ELISA was validated comparing sera from patients with histoplasmosis, related mycoses, and healthy individuals. The overall test specificity was 96%, with sensitivities of 100% (8/8) in acute disease, 90% (9/10) in chronic disease, 89% (8/9) in disseminated infection in individuals without HIV infection, 86% (12/14) in disseminated disease in the setting of HIV infection and 100% (3/3) in mediastinal histoplasmosis. These parameters are superior to the use of untreated histoplasmin in diagnostic ELISAs. The high specificities, sensitivities, and simplicity of our ELISA support further development of a deglycosylated HMIN ELISA for clinical use and for monitoring the humoral immune response during therapy in patients with chronic and disseminated histoplasmosis.

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Combinatorial epitope search: Pitfalls of library design

Cited by 14 publications

References 7 publications

Determination of Substrate Specificity for Peptide Deformylase through the Screening of a Combinatorial Peptide Library

Determination of Substrate Specificity for Peptide Deformylase through the Screening of a Combinatorial Peptide Library

II. Structure and specificity of the interaction between the FHA2 domain of rad53 and phosphotyrosyl peptides

Diagnostic and immunoprophylactic applications of synthetic peptides in veterinary microbiology

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