scholarly journals In vitro ribosome synthesis and evolution through ribosome display

2019 ◽  
Author(s):  
Michael J. Hammerling ◽  
Brian R. Fritz ◽  
Danielle J. Yoesep ◽  
Do Soon Kim ◽  
Erik D. Carlson ◽  
...  
Keyword(s):  
Cell Viability ◽  
Directed Evolution ◽  
Ribosome Display ◽  
Highly Active ◽  
Integrated Strategy ◽  
A Cell ◽  
Rapid Generation ◽  
Free Environment ◽  
Selection Of

ABSTRACTDirected evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

scholarly journals A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xun Chen ◽  
Matteo Gentili ◽  
Nir Hacohen ◽  
Aviv Regev
Keyword(s):  
In Vitro Selection ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Pseudotyped Virus ◽  
Receptor Binding Domain ◽  
Computational Pipeline ◽  
A Cell ◽  
Rapid Generation ◽  
Selection Of

AbstractAntibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.

scholarly journals Strain-Dependent Impact of G and SH Deletions Provide New Insights for Live-Attenuated HMPV Vaccine Development

Vaccines ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 164 ◽  
Author(s):  
Julia Dubois ◽  
Andrés Pizzorno ◽  
Marie-Hélène Cavanagh ◽  
Blandine Padey ◽  
Claire Nicolas de Lamballerie ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Specific Treatment ◽  
Respiratory Pathogen ◽  
Recombinant Viruses ◽  
Human Airway Epithelium ◽  
A Cell ◽  
Strain Dependent ◽  
Selection Of

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

scholarly journals Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Takehiro Nishikawa ◽  
Takeshi Sunami ◽  
Tomoaki Matsuura ◽  
Tetsuya Yomo
Keyword(s):  
Protein Synthesis ◽  
Directed Evolution ◽  
Single Copy ◽  
Functional Evaluation ◽  
Future Directions ◽  
Pure System ◽  
Protein Functions ◽  
A Cell ◽  
Evolution Of Proteins

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.

scholarly journals A cell-free framework for biological systems engineering

2015 ◽  
Author(s):  
Henrike Niederholtmeyer ◽  
Zachary Sun ◽  
Yutaka Hori ◽  
Enoch Yeung ◽  
Amanda Verpoorte ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Biological Systems ◽  
Biological Engineering ◽  
Free System ◽  
Node Negative ◽  
A Cell ◽  
Free Environment ◽  
Synthetic Networks

While complex dynamic biological networks control gene expression and metabolism in all living organisms, engineering comparable synthetic networks remains challenging1,2. Conducting extensive, quantitative and rapid characterization during the design and implementation process of synthetic networks is currently severely limited due to cumbersome molecular cloning and the difficulties associated with measuring parts, components and systems in cellular hosts. Engineering gene networks in a cell-free environment promises to be an efficient and effective approach to rapidly develop novel biological systems and understand their operating regimes3-5. However, it remains questionable whether complex synthetic networks behave similarly in cells and a cell-free environment, which is critical for in vitro approaches to be of significance to biological engineering. Here we show that synthetic dynamic networks can be readily implemented, characterized, and engineered in a cell-free framework and consequently transferred to cellular hosts. We implemented and characterized the “repressilator”6, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring these novel three-node and five-node oscillators to Escherichia coli, resulting in robust and synchronized oscillations reflecting the in vitro observation. We demonstrate that comprehensive circuit engineering can be performed in a cell-free system and that the in vitro results have direct applicability in vivo. Cell-free synthetic biology thus has the potential to drastically speed up design-build-test cycles in biological engineering and enable the quantitative characterization of synthetic and natural networks.

scholarly journals A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

2020 ◽  
Author(s):  
Xun Chen ◽  
Matteo Gentili ◽  
Nir Hacohen ◽  
Aviv Regev
Keyword(s):  
Neutralizing Antibodies ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Pseudotyped Virus ◽  
Free Antibody ◽  
A Cell ◽  
Heavy Chain Antibody ◽  
Rapid Generation ◽  
Unique Capability

AbstractAntibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

Evaluation of the Influence of Ferrite Magnetic Nanoparticle for Cancer Cell

2021 ◽  
Vol 323 ◽  
pp. 146-151
Author(s):  
Khishigdemberel Ikhbayar ◽  
Nomin Myagmar ◽  
Gantulga Davaakhuu ◽  
Uyanga Enkhnaran ◽  
Enkhmend Bekhbaatar ◽  
...  
Keyword(s):  
Cell Viability ◽  
Colony Formation ◽  
Magnetic Nanoparticle ◽  
In Vitro Cytotoxicity ◽  
Colony Formation Assay ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Viability Assay ◽  
A Cell

Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Mg 0.8 Ni 0.2 Fe 2 O 4 nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. HeLa cells exhibited cytotoxic effects when exposed to three different concentrations of 150 μg /ml, 100 μg /ml, and 50 μg /ml nanoparticles. Therefor e, c oncentrations of 50 μg /ml showed the lowest cytotoxic activity and the lowest toxicity to living cells. In vitro cytotoxicity of samples was then investigated by two methods, colony formation assay and cell viability assay. The Hela inhibited cell growth as 16.8% during heating by magnetic field generators.

scholarly journals Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo

2018 ◽  
Vol 109 (5) ◽  
pp. 1503-1512
Author(s):  
Shangke Huang ◽  
Lu Feng ◽  
Gaili An ◽  
Xiaojin Zhang ◽  
Zixuan Zhao ◽  
...  
Keyword(s):  
Single Chain ◽  
Ribosome Display ◽  
Single Chain Variable Fragments ◽  
Selection Of

scholarly journals Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 18
Author(s):  
Janne Leivo ◽  
Markus Vehniäinen ◽  
Urpo Lamminmäki
Keyword(s):  
In Vitro Selection ◽  
Specific Binding ◽  
Binding Properties ◽  
Phage Displays ◽  
Synthetic Antibody ◽  
Wide Range ◽  
Antibody Libraries ◽  
Rapid Generation ◽  
Selection Of

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

scholarly journals A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in anIn VitroCompartmentalized Cell-Free Translation System

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Atsushi Ogawa ◽  
Masayoshi Hayami ◽  
Shinsuke Sando ◽  
Yasuhiro Aoyama
Keyword(s):  
Stop Codon ◽  
Full Length ◽  
Translation System ◽  
Anticodon Loop ◽  
Ribosome Display ◽  
Amber Stop Codon ◽  
Free Translation ◽  
Translatable Mrna ◽  
Selection Of

Here is presented a concept forin vitroselection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNASer. Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNASer.

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