scholarly journals Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions

Molecules ◽  
2019 ◽  
Vol 24 (14) ◽  
pp. 2620 ◽  
Author(s):  
Soichiro Yamauchi ◽  
Yoshihiro Kobashigawa ◽  
Natsuki Fukuda ◽  
Manaka Teramoto ◽  
Yuya Toyota ◽  
...  
Keyword(s):  
High Speed ◽  
Genetic Manipulation ◽  
Low Cost ◽  
Dynamic Feature ◽  
Single Chain ◽  
Variable Regions ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Chain Fv ◽  
C Terminus

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression

2020 ◽  
Vol 168 (3) ◽  
pp. 257-263 ◽  
Author(s):  
Chenjiang Liu ◽  
Yoshihiro Kobashigawa ◽  
Soichiro Yamauchi ◽  
Natsuki Fukuda ◽  
Takashi Sato ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Low Cost ◽  
Recombinant Antibody ◽  
Expression System ◽  
Single Chain ◽  
Application Study ◽  
Variable Regions ◽  
Protein Ligation ◽  
Split Intein ◽  
Single Chain Fv

Abstract Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH–VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

scholarly journals Photocatalytic reduction of graphene oxide with cuprous oxide film under UV-vis irradiation

2020 ◽  
Vol 59 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Yao Wang ◽  
Jianqing Feng ◽  
Lihua Jin ◽  
Chengshan Li
Keyword(s):  
Graphene Oxide ◽  
Photoelectron Spectroscopy ◽  
Low Cost ◽  
Photocatalytic Reduction ◽  
Force Microscopy ◽  
Atomic Force ◽  
Reduced Graphene ◽  
Reduction Efficiency ◽  
Metal Organic ◽  
Reduction Effect

AbstractWe have grown Cu2O films by different routes including self-oxidation and metal-organic deposition (MOD). The reduction efficiency of Cu2O films on graphene oxide (GO) synthesized by modified Hummer’s method has been studied. Surface morphology and chemical state of as-prepared Cu2O film and GO sheets reduced at different conditions have also been investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). Results show that self-oxidation Cu2O film is more effective on phtocatalytic reduction of GO than MOD-Cu2O film. Moreover, reduction effect of self-oxidation Cu2O film to GO is comparable to that of environmental-friendly reducing agent of vitamin C. The present results offer a potentially eco-friendly and low-cost approach for the manufacture of reduced graphene oxide (RGO) by photocatalytic reduction.

scholarly journals Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Rina Hirano ◽  
Yasuhiro Arimura ◽  
Tomoya Kujirai ◽  
Mikihiro Shibata ◽  
Aya Okuda ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Dna Repair ◽  
High Speed ◽  
Histone Chaperones ◽  
Histone H2a ◽  
Force Microscopy ◽  
Atomic Force ◽  
Open Conformation ◽  
Replication Sites ◽  
Histone Exchange

AbstractH2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate “open conformation”, in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.

High-Speed Atomic Force Microscopy for Studying the Dynamic Behavior of Protein Molecules at Work

2006 ◽  
Vol 45 (3B) ◽  
pp. 1897-1903 ◽  
Author(s):  
Toshio Ando ◽  
Takayuki Uchihashi ◽  
Noriyuki Kodera ◽  
Atsushi Miyagi ◽  
Ryo Nakakita ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Dynamic Behavior ◽  
High Speed ◽  
Force Microscopy ◽  
Atomic Force ◽  
Protein Molecules
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