Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris

2021 ◽  
pp. 151-167
Author(s):  
Laia Montoliu-Gaya ◽  
Sandra Villegas
Keyword(s):  
Pichia Pastoris ◽  
Single Chain ◽  
Single Chain Variable Fragments

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display

1994 ◽  
Vol 269 (13) ◽  
pp. 9533-9538
Author(s):  
S.J. Deng ◽  
C.R. MacKenzie ◽  
J. Sadowska ◽  
J. Michniewicz ◽  
N.M. Young ◽  
...  
Keyword(s):  
Phage Display ◽  
Carbohydrate Binding ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Selection Of

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

scholarly journals Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Kshitij Wagh ◽  
Penny L. Moore ◽  
Lynn Morris
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Neutralization Assay ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
High Potency ◽  
Hill Model ◽  
Mucosal Tissues ◽  
Action Model ◽  
Single Chain Variable Fragments

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1μg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

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