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 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.

Construction and Expression of a Bifunctional Single-Chain Antibody against Bacillus cereusSpores

1998 ◽  
Vol 64 (7) ◽  
pp. 2490-2496 ◽  
Author(s):  
Kai Koo ◽  
Peggy M. Foegeding ◽  
Harold E. Swaisgood
Keyword(s):  
Monoclonal Antibody ◽  
Recombinant Antibody ◽  
Expression System ◽  
Variable Region ◽  
Hybridoma Cells ◽  
Antigen Specificity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Western Blots ◽  
Variable Region Genes

ABSTRACT The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

scholarly journals Virus Strain Discrimination Using Recombinant Antibodies

2000 ◽  
Vol 16 (1-2) ◽  
pp. 95-97 ◽  
Author(s):  
N. Boonham ◽  
I. Barker
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Recombinant Antibody ◽  
Plant Viruses ◽  
Antibody Fragments ◽  
Single Chain ◽  
Traditional Methods ◽  
Assay Sensitivity ◽  
Single Chain Fv ◽  
Selection Of

Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological facilities. Single chain Fv antibody fragments (scFv) have been selected from a synthetic phage-antibody library by affinity selection with purifiedPotato virus Y, ordinary strain (PVYO). The scFv selected was specific for PVY and detected 7 out of 9 isolates of PVYOwhilst it did not detect 15 isolates from the closely related necrotic strains PVYNand PVYNTN. In ELISA the scFv could be used to detect virus at concentrations of 50 ng/ml in plant sap and was shown to have similar limits of detection as commercially available PVY monoclonal antibodies. These results highlight the potential of the technology for the selection of strain specific antibodies with an affinity and assay sensitivity similar to traditional monoclonal antibodies and their use in viral diagnostics.

scholarly journals Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

2015 ◽  
Vol 112 (32) ◽  
pp. 9860-9865 ◽  
Author(s):  
Changming Yu ◽  
Gennett M. Pike ◽  
Tommy A. Rinkoski ◽  
Cristina Correia ◽  
Scott H. Kaufmann ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
Protein Translation ◽  
Avian Leukosis Virus ◽  
Eukaryotic Expression ◽  
Eukaryotic Cells ◽  
Single Chain ◽  
Mammalian Expression ◽  
Single Chain Fv ◽  
Fv Antibody

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

scholarly journals Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3393-3399 ◽  
Author(s):  
Gregory Kanter ◽  
Junhao Yang ◽  
Alexei Voloshin ◽  
Shoshana Levy ◽  
James R. Swartz ◽  
...  
Keyword(s):  
B Cell ◽  
Expression System ◽  
Specific Antigen ◽  
Therapeutic Vaccine ◽  
Antibody Fragment ◽  
B Cell Lymphoma ◽  
Keyhole Limpet Hemocyanin ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody

Abstract The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli—based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

scholarly journals Generation and Characterization of a Spike Glycoprotein Domain A-Specific Neutralizing Single-Chain Variable Fragment against Porcine Epidemic Diarrhea Virus

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 833
Author(s):  
Chia-Yu Chang ◽  
Yong-Sheng Wang ◽  
Jou-Fei Wu ◽  
Tzu-Jing Yang ◽  
Yen-Chen Chang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Expression System ◽  
Single Chain ◽  
Variable Regions ◽  
Diarrhea Virus ◽  
Single Chain Fragment Variable ◽  
Neutralizing Monoclonal Antibody ◽  
Porcine Epidemic Diarrhea ◽  
Lactogenic Immunity

The emergence of the genotype (G) 2 and re-emergence of the G1 porcine epidemic diarrhea virus (PEDV) has caused severe economic impacts in the past decade. Developments of efficient vaccines against new variants of PEDV have been challenging, not least because of the difficulties in eliciting mucosal and lactogenic immunity. A single-chain fragment variable (scFv) capable of efficient antigen recognition is an alternative to vaccination and treatment of a viral infection. In the present study, the variable regions of the light chain and the heavy chain of a G2b PEDV spike domain A (S1A)-specific neutralizing monoclonal antibody (mAb) were sequenced, constructed with a (G4S) x3 linker, and produced by a mammalian protein expression system. Our results demonstrated that the PEDV S1A domain scFv was able to bind to S proteins of both G1 and G2b PEDVs. Nevertheless, the scFv was only capable of neutralizing the homologous G2b PEDV but not the G1 PEDV. The binding ability of the G2b-specific neutralizing scFv was not able to predict the neutralizing ability toward heterologous PEDV. The anti-PEDV S1A scFv presented herein serves as a potential therapeutic candidate against the virulent G2b PEDV.

scholarly journals Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Shukra M. Aavula ◽  
Sridevi V. Nimmagadda ◽  
Neelakantam Biradhar ◽  
Samuel Sula ◽  
Dev Chandran ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Specific Binding ◽  
Recombinant Antibody ◽  
Antigenic Site ◽  
Antibody Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Single Chain Fv ◽  
Rabies Glycoprotein

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

scholarly journals Construction of a Functional Single-Chain Variable Fragment Antibody against Hemagglutinin from Porphyromonas gingivalis

1998 ◽  
Vol 66 (5) ◽  
pp. 2207-2212 ◽  
Author(s):  
Yasuko Shibata ◽  
Kimiyo Kurihara ◽  
Hisashi Takiguchi ◽  
Yoshimitsu Abiko
Keyword(s):  
Porphyromonas Gingivalis ◽  
Recombinant Antibody ◽  
Expression System ◽  
Passive Immunization ◽  
Amino Acid Sequences ◽  
Hemagglutinating Activity ◽  
Bacterial Invasion ◽  
Nucleotide Sequencing ◽  
Single Chain Variable Fragment ◽  
Single Chain

ABSTRACT Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against theP. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791–2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalisvesicles. In addition, the ScFv antibody inhibited theP. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vκ12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.

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