Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role.

Phage Display Preparation of Specific Polypeptides in Atherosclerotic Foam Cells

Atherosclerosis and related complications are the most common causes of death in modern societies. Macrophage-derived foam cells play critical roles in the initiation and progression of atherosclerosis. Effective, rapid, and instrument-independent detection in the early stage of chronic atherosclerosis progression could provide an opportunity for early intervention and treatment. Therefore, as a starting point, in this study, we aimed to isolate and prepare foam cell-specific polypeptides using a phage display platform. The six target polypeptides, which were acquired in this study, were evaluated by ELISA and showed strong specificity with foam cells. Streptavidin coupled quantum dots (QDs) were used as fluorescence developing agents, and images of biotin-modified polypeptides specifically binding with foam cells were clearly observed. The polypeptides obtained in this study could lay the foundation for developing a rapid detection kit for early atherosclerosis lesions and could provide new materials for research on the mechanisms of foam cell formation and the development of blocking drugs.

Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

Reversal of pancreatic desmoplasia by a tumour stroma-targeted nitric oxide nanogel overcomes TRAIL resistance in pancreatic tumours

ObjectiveStromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC.DesignNitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro–in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo.ResultsThe delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy.ConclusionThe co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.

Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively.

Phage display – a tool for detection and prevention against pathogens. A review

In this article are reviewed the promising uses of phage display in the areas such as microbial pathogens detection of and vaccination. Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phages thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but have also the capability to replicate. Recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.