Site directed biotinylation of filamentous phage structural proteins

Filamentous bacteriophages have been used in numerous applications for the display of antibodies and random peptide libraries. Here we describe the introduction of a 13 amino acid sequence LASIFEAQKIEWR (designated BT, which is biotinylated in vivo by E. coli) into the N termini of four of the five structural proteins of the filamentous bacteriophage fd (Proteins 3, 7, 8 and 9). The in vivo and in vitro biotinylation of the various phages were compared. The production of multifunctional phages and their application as affinity reagents are demonstrated

Mapping and molecular characterization of novel monoclonal antibodies to conformational epitopes on NH2 and COOH termini of mammalian tryptophanyl-tRNA synthetase reveal link of the epitopes to aggregation and Alzheimer's disease

Tryptophanyl-tRNA synthetase (TrpRS) is an interferon-induced phosphoprotein with autoantigenic and cytokine activities detected in addition to its canonical function in tRNA aminoacylation. The availability of monoclonal antibodies (mAbs) specific for TrpRS is important for development of tools for TrpRS monitoring. A molecular characterization of two mAbs raised in mice, using purified, enzymatically active bovine TrpRS as the inoculating antigen, is presented in this report. These IgG1 antibodies are specific for bovine, human and rabbit but not E. coli TrpRS. Immunoreactivity and specificity of mAbs were verified with purified recombinant hTrpRS expressed in E. coli and TrpRS-derived synthetic peptides. One of the mAbs, 9D7 is able to disaggregate fibrils formed by Ser32-Tyr50 TrpRS-peptide. Epitope mapping revealed that disaggregation ability correlates with binding of 9D7 to this peptide in ELISA and immunocytochemistry. This epitope covers a significant part of N-terminal extension that suggested to be proteolytically deleted in vivo from the full-length TrpRS whereas remaining COOH-fragment possesses a cytokine activity. For epitope mapping of mAb 6C10, the affinity selected phage-displayed peptides were used as a database for prediction of conformational discontinuous epitopes within hTrpRS crystal structure. Using computer algorithm, this epitope is attributed to COOH-terminal residues Asp409-Met425. In immunoblotting, the 6C10 mAb reacts preferably with (i) oligomer than monomer, and (ii) bound than free TrpRS forms. The hTrpRS expression was shown to correlate with growth rates of neuroblastoma and pancreatic cancer cells. Immunohistochemically both mAbs revealed extracellular plaque-like aggregates in hippocampus of Alzheimer's disease brain

Deep Panning: Steps towards Probing the IgOme

Background: Polyclonal serum consists of vast collections of antibodies, products of differentiated B-cells. The spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. The complete repertoire of antibody specificities in blood, the IgOme, is therefore an extraordinarily rich source of information–a molecular record of previous encounters as well as a status report of current immune activity. The ability to profile antibody specificities of polyclonal serum at exceptionally high resolution has been an important and serious challenge which can now be overcome. Methodology/Principal Findings: Here we illustrate the application of Deep Panning, a method that combines the flexibility of combinatorial phage display of random peptides with the power of high-throughput deep sequencing. Deep Panning is first applied to evaluate the quality and diversity of naïve random peptide libraries. The production of very large data sets, hundreds of thousands of peptides, has revealed unexpected properties of combinatorial random peptide libraries and indicates correctives to ensure the quality of the libraries generated. Next, Deep Panning is used to analyze a model monoclonal antibody in addition to allowing one to follow the dynamics of biopanning and peptide selection. Finally Deep Panning is applied to profile polyclonal sera derived from HIV infected individuals. Conclusions/Significance: The ability to generate and characterize hundreds of thousands of affinity-selected peptide

Pie charts depicting the proportion of unique peptides in phage display libraries.

<p>A total of 155,241 inserts were read for the random phage display peptide library (<b>A</b>). 24% of the peptides contained at least one UAA or UGA stop codon (red plus dark red). 58% of the peptides were unique containing a UAG stop codon (light green) of these some exist in multiple copies (3% of the total, dark green). 15% of the peptides were completely devoid of stop codons (blue, less than 1% had 2–5 copies). Pie Chart (<b>B</b>) depicts the same set of peptides devoid of all those that had detectable frameshifted inserts (37,223 inserts leaving 118,018 functional peptides of which ca 1% contained stop codons UAA and UGA nonetheless). A second library was constructed in DH5alpha <i>supE144</i> cells (<b>C</b>). Values below 1% are not given.</p

Three rounds of panning with mAb GV4H3.

<p>GV4H3 mAb was used to bio-pan the 7 mer random peptide library 3 consecutive rounds of panning (Capture #1 through #3) and compared with the naïve library. For each sample the 20 top most frequent peptides are given along with the number of times they appear. The number of unique versus total peptides is shown as well. Numbers in parentheses represent the percent value of the total peptides for each category. Bold sequences indicate peptides that are carried over from Capture #1. Bold and Italic sequences indicate peptides carried over from Capture #2.</p

BLASTP analysis of HIVIG-captured peptides against viral coat proteins.

<p>Of the 223 top unique HIVIG-captured peptides, 18 (8%) scored hits in BLASTP analysis against the HIV-1_HXB2 gp160 (blue). Repeating this procedure with the same protein but scrambled gives an average value of 5.5 hits when performed 1,000 times (2.5%±2.2 s.d., red). The differences between native and scrambled coat proteins BLASTP results of 11 other RNA viruses were not found to be significant. <i>*P</i><0.01.</p

Deep Panning with mAb GV4H3.

<p>(<b>A</b>) Mapitope prediction of the GV4H3 epitope on HIV gp120. The top 20 peptides of Capture #2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) were used as the dataset for Mapitope prediction of the GV4H3 epitope. The single predicted cluster comprises two discontinuous segments of the antigen (green and blue) brought to flank the core of the epitope (residues 221–226, pink). (<b>B-E</b>) MEME analysis of the GV4H3 derived peptides. The 20 top most frequent peptides of Capture #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) generated a major motif “AGWAV”. This motif (<b>B</b>) and three additional motifs are identified when all 4,823 peptides are analyzed. The “VGF” motif (<b>C</b>) is a simpler version of the major motif. The two additional minor motifs (<b>D</b> and <b>E</b>) do not have obvious similarity to the epitope of the mAb. The “ADGIGGG” motif clearly corresponds with the most frequent peptide ADGIVGW (see text). The numbers in red represent the number of unique peptides that define each motif.</p