scholarly journals A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam Chin-Fatt ◽  
Reza Saberianfar ◽  
Rima Menassa
Keyword(s):  
Disulfide Bonds ◽  
Rational Design ◽  
De Novo ◽  
Passive Immunization ◽  
Fold Increase ◽  
Secretory Component ◽  
Secretory Iga ◽  
In Planta ◽  
Design Strategies ◽  
Single Domain Antibody

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

scholarly journals A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam Chin-Fatt ◽  
Rima Menassa
Keyword(s):  
Fusion Proteins ◽  
De Novo ◽  
Molecular Farming ◽  
Passive Immunization ◽  
Limiting Factors ◽  
Expression Vectors ◽  
In Planta ◽  
Single Domain Antibody ◽  
Oxidative Folding ◽  
Thylakoid Lumen

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

scholarly journals Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

2021 ◽  
Author(s):  
Ping Che ◽  
Emily Wu ◽  
Marissa K. Simon ◽  
Ajith Anand ◽  
Keith Lowe ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Genome Editing ◽  
De Novo ◽  
Callus Formation ◽  
Fold Increase ◽  
Control Treatment ◽  
In Planta ◽  
Culture Methods ◽  
Agrobacterium Mediated Transformation ◽  
Sorghum Genotypes

For many important crops including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.

scholarly journals Oral Immunotherapy With Human Secretory Immunoglobulin A Improves Survival in the Hamster Model of Clostridioides difficile Infection

2021 ◽  
Author(s):  
Estelle F Chiari ◽  
William Weiss ◽  
Michael R Simon ◽  
Stephan T Kiessig ◽  
Mark Pulse ◽  
...  
Keyword(s):  
Immunoglobulin A ◽  
Passive Immunization ◽  
Secretory Component ◽  
Oral Immunotherapy ◽  
Secretory Iga ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Secretory Immunoglobulin ◽  
Donor Plasma

Abstract Coadministration of human secretory IgA (sIgA) together with subtherapeutic vancomycin enhanced survival in the Clostridioides difficile infection (CDI) hamster model. Vancomycin (5 or 10 mg/kg × 5 days) plus healthy donor plasma sIgA/monomeric IgA (TID × 21 days) or hyperimmune sIgA/monomeric IgA (BID × 13 days) enhanced survival. Survival was improved compared to vancomycin alone, P = .018 and .039 by log-rank Mantel-Cox, for healthy and hyperimmune sIgA, respectively. Passive immunization with sIgA (recombinant human secretory component plus IgA dimer/polymer from pooled human plasma) can be administered orally and prevents death in a partially treated CDI hamster model.

scholarly journals General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shay Laps ◽  
Fatima Atamleh ◽  
Guy Kamnesky ◽  
Hao Sun ◽  
Ashraf Brik
Keyword(s):  
Drug Discovery ◽  
Disulfide Bonds ◽  
Unsolved Problem ◽  
De Novo ◽  
Therapeutic Potential ◽  
Selective Activation ◽  
One Pot ◽  
Synthetic Strategy ◽  
The One ◽  
Game Changer

AbstractDespite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

scholarly journals Computational and Rational Design of Single-Chain Antibody against Tick-Borne Encephalitis Virus for Modifying Its Specificity

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1494
Author(s):  
Ivan K. Baykov ◽  
Pavel Y. Desyukevich ◽  
Ekaterina E. Mikhaylova ◽  
Olga M. Kurchenko ◽  
Nina V. Tikunova
Keyword(s):  
Rational Design ◽  
Fold Increase ◽  
Encephalitis Virus ◽  
Chimeric Antibody ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Glycoprotein E ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Far Eastern

Tick-borne encephalitis virus (TBEV) causes 5−7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

scholarly journals Efficient In Planta Detection and Dissection of De Novo Mutation Events in theArabidopsis thalianaDisease Resistance GeneUNI

2016 ◽  
Vol 57 (6) ◽  
pp. 1123-1132
Author(s):  
Tomohiko Ogawa ◽  
Akiko Mori ◽  
Kadunari Igari ◽  
Miyo Terao Morita ◽  
Masao Tasaka ◽  
...  
Keyword(s):  
De Novo ◽  
De Novo Mutation ◽  
In Planta

Design of peptide affinity ligands for S-protein: a comparison of combinatorial and de novo design strategies

2013 ◽  
Vol 17 (2) ◽  
pp. 357-369 ◽  
Author(s):  
Divya Chandra ◽  
Christopher J. Morrison ◽  
James Woo ◽  
Steven Cramer ◽  
Pankaj Karande
Keyword(s):  
De Novo ◽  
Protein A ◽  
De Novo Design ◽  
Design Strategies ◽  
S Protein ◽  
Affinity Ligands ◽  
Peptide Affinity

scholarly journals Polymeric micelles and molecular modeling applied to the development of radiopharmaceuticals

2012 ◽  
Vol 48 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Sibila Roberta Marques Grallert ◽  
Carlota de Oliveira Rangel-Yagui ◽  
Kerly Fernanda Mesquita Pasqualoto ◽  
Leoberto Costa Tavares
Keyword(s):  
Molecular Modeling ◽  
Rational Design ◽  
Polymeric Micelles ◽  
Single Photon ◽  
Photon Emission ◽  
Disease Process ◽  
Diagnostic Techniques ◽  
Emission Computed Tomography ◽  
Amphiphilic Copolymers ◽  
Design Strategies

Micelles composed of amphiphilic copolymers linked to a radioactive element are used in nuclear medicine predominantly as a diagnostic application. A relevant advantage of polymeric micelles in aqueous solution is their resulting particle size, which can vary from 10 to 100 nm in diameter. In this review, polymeric micelles labeled with radioisotopes including technetium (99mTc) and indium (111In), and their clinical applications for several diagnostic techniques, such as single photon emission computed tomography (SPECT), gamma-scintigraphy, and nuclear magnetic resonance (NMR), were discussed. Also, micelle use primarily for the diagnosis of lymphatic ducts and sentinel lymph nodes received special attention. Notably, the employment of these diagnostic techniques can be considered a significant tool for functionally exploring body systems as well as investigating molecular pathways involved in the disease process. The use of molecular modeling methodologies and computer-aided drug design strategies can also yield valuable information for the rational design and development of novel radiopharmaceuticals.

scholarly journals Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

2021 ◽  
Vol 8 ◽  
Author(s):  
Han Zhang ◽  
Lei Wang ◽  
Xiang Yang ◽  
Zhiwei Lian ◽  
Yinbin Qiu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Prokaryotic Expression ◽  
Disulfide Bonds ◽  
De Novo ◽  
Animal Experiments ◽  
Cone Snail ◽  
Active Peptides ◽  
Neuroscience Research ◽  
First Time ◽  
Mass Spectrometry Mass Spectrometry

Conopeptides from the marine cone snails are a mixture of cysteine-rich active peptides, representing a unique and fertile resource for neuroscience research and drug discovery. The ConoServer database includes 8,134 conopeptides from 122 Conus species, yet many more natural conopeptides remain to be discovered. Here, we identified 517 distinct conopeptide precursors in Conus quercinus using de novo deep transcriptome sequencing. Ten of these precursors were verified at the protein level using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The combined gene and protein analyses revealed two novel gene superfamilies (Que-MNCLQ and Que-MAMNV), and three other gene superfamilies (N, P, and I1) were reported for the first time in C. quercinus. From the Que-MAMNV superfamily, a novel conotoxin, Que-0.1, was obtained via cloning and prokaryotic expression. We also documented a new purification process that can be used to induce the expression of conopeptides containing multiple pairs of disulfide bonds. The animal experiments showed that Que-0.1 strongly inhibited neuroconduction; the effects of Que-1.0 were 6.25 times stronger than those of pethidine hydrochloride. In addition, a new cysteine framework (CC-C-C-C-C-C-CC-C-C-C-C-C) was found in C. quercinus. These discoveries accelerate our understanding of conopeptide diversity in the genus, Conus and supply promising materials for medical research.

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