Selection and characterization of high affinity VEGFR1 antibodies from a novel human binary code scFv phage library

VEGFR1 is a receptor tyrosine kinase that has been implicated in cancer pathogenesis. It is upregulated in angiogenic endothelial cells and expressed on human tumor cells as well. VEGFR1 positive hematopoietic progenitor cells home to sites of distant metastases prior to the arrival of the tumor cells thus establishing a pre-metastatic niche. To discover high affinity human antibodies selective for VEGFR1 molecular imaging or for molecularly targeted therapy, a novel phage display scFv library was assembled and characterized. The library was constructed from the humanized 4D5 framework that was mostly comprised tyrosine and serine residues in four complimentarity determining regions (CDRs). The library produced diverse and functional antibodies against a panel of proteins, some of which are of biomedical interest including, CD44, VEGFA, and VEGFR1. After panning, these antibodies had affinity strong enough for molecular imaging or targeted drug delivery without the need for affinity maturation. One of the anti-VEGFR1 scFvs recognized its cognate receptor and was selective for the VEGFR1.     

Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library

To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb2-LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples.     

Phage display: an important tool in the discovery of peptides with anti-HIV activity

Human immunodeficiency virus (HIV) remains a worldwide health problem despite huge investments and research breakthroughs, and no single drug is effective in killing the virus yet. Among new strategies to control HIV infection, the phage display (PD) technology has become a promising tool in the discovery of peptides that can be used as new drugs, or also as possible vaccine candidates. This review discusses basic aspects of PD and its use to advance two main objectives related to combating HIV-1 infection: the identification of peptides that inhibit virus replication and the identification of peptides that induce the production of neutralizing antibodies. We will cover the different approaches used for mapping and selection of mimotopes, and discuss the promising results of these biologicals as antiviral agents.     

Tagged polymerase chain reaction subtractive hybridization for the enrichment of phage display random peptide libraries

Affinity selection of phage display peptide libraries is routinely used for isolating peptides capable of binding a range of molecules, including antibodies and receptors. This process is most successful when the selecting molecule is relatively pure, for example, a monoclonal antibody. However, isolation of peptides able to bind to target molecules present in a complex mixture is more difficult because the affinity selection process isolates peptides capable of binding to all molecules present in the mixture. Here we describe the development of a tagged polymerase chain reaction (PCR) subtractive hybridization method that is universally applicable for the targeted isolation of peptides able to bind to unique molecules within a complex mixture. We also describe a discriminatory limiting dilution PCR method that can be used to optimize hybridization conditions.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

A large library based on a novel (CH2) scaffold: Identification of HIV-1 inhibitors

Isolated immunoglobulin CH2 domains were proposed as scaffolds for selection of binders with potential effector functions. We tested the feasibility of this approach by constructing a large (size 5 × 10¹⁰) library where all amino acids in two loops (BC and FG) were mutated to four residues (Y, A, D, or S). Three binders were selected from this library by panning against a gp120-CD4 complex. The strongest binder, m1a1, recognized specifically a highly conserved CD4i epitope and inhibited to various extents seven out of nine HIV-1 isolates from different clades. The loop BC and the conformational state of the scaffold are critical for its binding. These results provide a proof of concept for the potential of CH2 as a scaffold for construction of libraries containing potentially useful binders. The newly identified HIV-1 inhibitors could be further improved to candidate therapeutics and/or used as research reagents for exploration of conserved gp120 structures.     

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.     

Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells

The ICAM-1 adhesion molecule is expressed selectively at low levels on endothelial cells but is strongly upregulated in dysfunctional endothelial cells associated with inflammation, cancer, and atherogenesis. Using COS-7 cells transfected with murine ICAM-1 (mICAM-1) as a target receptor, a phage display library was screened. Clones were selected by elution with a mAb specific for a functional epitope of ICAM-1 and a novel peptide sequence binding to the extracellular domain of mICAM-1 was identified that can potentially be used as a targeting vector aimed at dysfunctional endothelium. We further showed that the targeting specificity of the peptide was retained following its incorporation at the N terminal end of a large chimeric protein. Moreover, this chimeric protein containing the mICAM-1-specific sequence was found to inhibit ICAM-1-mediated intercellular adhesion during antigen presentation. Taken together, these results demonstrate the potential for improving the cell-selectivity and properties of therapeutical agents toward targeting adhesion molecules involved in cell-cell interactions.     

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 “QPTKDSSPPLRV” and NO4 “STTSPPAVPHNN” peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity.     

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil

Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC₅₀) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml⁻¹, respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules.     

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 dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in 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-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion 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. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

Identification of the HIV-1 gp41 core-binding motif--HXXNPF

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.     

The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.     

Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein

The Nef proteins of simian and human immunodeficiency viruses are known to directly bind and downregulate the CD4 receptor of infected cells. Recent results suggest that residues forming an alpha-helix N-cap in the CD4 cytoplasmic domain play a role in binding of CD4 to human immunodeficiency virus type 1 Nef protein. We determined the dissociation constants between Nef and several CD4 peptides that contain or do not contain the respective alpha-helix N-cap. Further, we compared helical secondary structure content of these CD4 peptide variants by circular dichroism spectroscopy. We conclude that presence of an alpha-helix in CD4 cytoplasmic domain increases CD4 affinity to Nef. In addition, the amino acid sequence of residues forming the helix N-cap influences CD4 affinity to Nef, too. Finally, the structural changes induced in Nef and CD4 upon binding to each other are investigated.     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1(MOC1)gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering.we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins(MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 resulted in enhanced tillering and reduced plant height.In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering.     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering,we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 res...     

Phage display combinatorial libraries of short peptides: ligand selection for protein purification

A library of heptapeptides displayed on the surface of filamentous phage M13 was evaluated as a potential source of affinity ligands for the purification of Rhizomucor miehei lipase. Two independent selection (biopanning) protocols were employed: the enzyme was either physically adsorbed on polystyrene or chemically immobilized on small magnetic beads. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichment of the library with “doublet” clones i.e. the phage species which carried two consecutive sequences of heptapeptides, whilst no such clones were observed from the screening using lipase attached to magnetic beads. The binding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best “monomeric” clone did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. The scope of application of this methodology and the possibility of preparing phage-based affinity materials are briefly discussed.