scholarly journals Human Monoclonal scFvs that Neutralize Fribrinogenolytic Activity of Kaouthiagin, a Zinc-Metalloproteinase in Cobra (Naja kaouthia) Venom

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 509
Author(s):  
Jirawat Khanongnoi ◽  
Siratcha Phanthong ◽  
Onrapak Reamtong ◽  
Anchalee Tungtronchitr ◽  
Wanpen Chaicumpa ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Phage Display Library ◽  
Single Step ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Venom Component ◽  
E Coli ◽  
Naja Kaouthia

Snake venom-metalloproteinases (SVMPs) are the primary factors that disturb hemostasis and cause hemorrhage in the venomous snake bitten subjects. Kaouthiagin is a unique SVMP that binds and cleaves von Willebrand factor (vWF) at a specific peptide bond leading to inhibition of platelet aggregation, which enhances the hemorrhage. Kaouthiagin is a low abundant venom component of Thai cobra (Naja kaouthia); thus, most horse-derived antivenins used for cobra bite treatment do not contain adequate anti-kaouthiagin. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with kaouthiagin activity for further clinical use. Kaouthiagin was purified from N. kaouthia-holovenom by a single-step gel-filtration chromatography. The purified venom component was used in phage-biopanning to select the kaouthiagin-bound HuscFv-displayed-phage clones from a HuscFv-phage display library. The selected phages were used to infect Escherichia coli bacteria. Soluble HuscFvs expressed by three phage-transformed-E. coli clones interfered with cobra kaouthiagin binding to human vWF. Computerized simulation indicated that HuscFv of two phage-transformed E. coli clones formed contact interface with kaouthiagin residues at or near catalytic site and effectively inhibited fibrinogenolytic activity of the kaouthiagin. The HuscFvs have therapeutic potential as an adjunct of antivenins in treatment of bleeding caused by venomous snakebites.

scholarly journals Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2201
Author(s):  
Tyng Hwey Tan ◽  
Elizabeth Patton ◽  
Carol A. Munro ◽  
Dora E. Corzo-Leon ◽  
Andrew J. Porter ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Transmembrane Protein ◽  
Fluorescent Microscopy ◽  
Phage Display Library ◽  
Inflammasome Activation ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Surface Plasmon Resonance Analysis ◽  
Antibody Phage ◽  
Antibody Phage Display

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.

scholarly journals Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Hussin A. Rothan ◽  
Ammar Y. Abdulrahman ◽  
Pottayil G. Sasikumer ◽  
Shatrah Othman ◽  
Noorsaadah Abd Rahman ◽  
...  
Keyword(s):  
Viral Replication ◽  
Therapeutic Potential ◽  
Solid Phase ◽  
Dengue Infection ◽  
Solid Phase Peptide Synthesis ◽  
Single Chain ◽  
E Coli ◽  
Social Burden ◽  
Disulphide Bonds ◽  
Starting Point

Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein inE. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC50of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P<0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P<0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.

scholarly journals Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library

Leukemia ◽  
1998 ◽  
Vol 12 (8) ◽  
pp. 1295-1302 ◽  
Author(s):  
PMW Willems ◽  
RMA Hoet ◽  
ELPG Huys ◽  
JMH Raats ◽  
EJBM Mensink ◽  
...  
Keyword(s):  
Phage Display ◽  
Plasma Cells ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Single Chain ◽  
Specific Detection ◽  
Single Chain Antibody

scholarly journals Selection and characterization of scFv antibody against nucleocapsid protein of Porcine reproductive and respiratory syndrome virus

2020 ◽  
Vol 89 (1) ◽  
pp. 39-45
Author(s):  
Magdalena Krasna ◽  
Vladimír Celer
Keyword(s):  
Infectious Agent ◽  
Scfv Antibody ◽  
Respiratory Syndrome Virus ◽  
Single Chain ◽  
Single Chain Antibody ◽  
N Protein ◽  
E Coli ◽  
Metal Affinity ◽  
Binding Epitope

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread infectious agent in pigs. Nucleocapsid (N) protein of PRRSV has been identified as the most immunodominant viral protein. The main goal of the work was the selection and characterization of a single-chain antibody fragments (scFv) antibody specific to the N protein. Specific scFv antibody clone D5 was selected from the Tomlinson phagemid library and purified by immobilized metal affinity chromatography from the periplasmatic space of E. coli cells. The antibody was then characterized by sequencing and the ability to recognize the native virus N protein by Western blot and competitive ELISA. Pepscan analysis identified the position of the binding epitope between amino acids 62–84 of the N protein. Our study could help to improve the diagnostics and prevention of PRRSV in Central Europe.

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification

2004 ◽  
Vol 42 (3) ◽  
Author(s):  
Andreas Ritsch ◽  
Christoph Ebenbichler ◽  
Elisabeth Naschberger ◽  
Wilfried Schgoer ◽  
Ursula Stanzl ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Cholesteryl Ester Transfer Protein ◽  
Density Lipoprotein ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Recombinant Phage ◽  
Transfer Protein ◽  
High Affinity

AbstractCholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), bothknown to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective K

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