scholarly journals High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures

BioTechniques ◽  
2021 ◽  
Author(s):  
Yoshiro Hanyu ◽  
Mieko Kato
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Recombinant Antibody ◽  
Antibody Fragments ◽  
High Yield ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Antibody Fragments ◽  
Single Chain Variable Fragments ◽  
High Yields

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

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 Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0131484 ◽  
Author(s):  
Christiane Y. Ozaki ◽  
Caio R. F. Silveira ◽  
Fernanda B. Andrade ◽  
Roberto Nepomuceno ◽  
Anderson Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Chain ◽  
E Coli ◽  
Heat Stable ◽  
Heat Labile ◽  
Single Chain Variable Fragments

scholarly journals Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120481 ◽  
Author(s):  
Daniela Luz ◽  
Gang Chen ◽  
Andrea Q. Maranhão ◽  
Leticia B. Rocha ◽  
Sachdev Sidhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Recombinant Antibody ◽  
Antibody Fragments ◽  
Recombinant Antibody Fragments

scholarly journals Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241773
Author(s):  
Vijay Gupta ◽  
Indulekha P. Sudhakaran ◽  
Zeyaul Islam ◽  
Nishant N. Vaikath ◽  
Issam Hmila ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Recombinant Antibody ◽  
Structural Alignment ◽  
Lewy Bodies ◽  
High Specificity ◽  
Binding Pocket ◽  
Antibody Fragments ◽  
Structural Topology ◽  
Single Chain ◽  
Recombinant Antibody Fragments

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies

2020 ◽  
Author(s):  
Vijay Gupta ◽  
Indulekha P. Sudhakaran ◽  
Zeyaul Islam ◽  
Nishant N. Vaikath ◽  
Issam Hmila ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Recombinant Antibody ◽  
Structural Alignment ◽  
Lewy Bodies ◽  
High Specificity ◽  
Binding Pocket ◽  
Antibody Fragments ◽  
Structural Topology ◽  
Single Chain ◽  
Recombinant Antibody Fragments

Abstract Background: Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. Results: In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E.coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. Conclusion: The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

scholarly journals Soluble Expression of Small Antibody Fragments against PD-L1 Using Escherichia coli with High Yield and Purity

2021 ◽  
Vol 11 (19) ◽  
pp. 9149
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Escherichia Coli ◽  
Tumor Cells ◽  
Surface Protein ◽  
Antibody Fragments ◽  
High Yield ◽  
Soluble Form ◽  
Single Chain ◽  
High Penetration ◽  
Variable Heavy ◽  
Variable Light

Programmed death-ligand 1 (PD-L1) is a surface protein overexpressed in tumor cells. Recently, targeted therapy using PD-L1 antibodies to reconstitute the antitumor activity of T cells has received considerable attention as a cancer treatment. Among the several types of anti-PD-L1 antibodies, small-sized antibody fragments are useful agents to block PD-L1 for experimental and therapeutic purposes owing to their high penetration efficacy toward dense tumor cells. Herein, we expressed and purified recombinant single chain fragment of variable domain, variable heavy chain, and variable light chain, against PD-L1 in a soluble form using Escherichia coli, resulting in their high yield and high purity. We confirmed the antigen-binding efficiency of these antibody fragments, which showed antigen concentration-dependent responses. These results suggest that these small antibody fragments can serve as new agents for blocking or detecting PD-L1.

scholarly journals Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

2019 ◽  
Vol 41 (1) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Expression Vector ◽  
Recombinant Antibody ◽  
Soluble Form ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Insoluble Form ◽  
Single Chain Variable Fragments

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3343-3349 ◽  
Author(s):  
William J. J. Finlay ◽  
Iain Shaw ◽  
Joanna P. Reilly ◽  
Marian Kane
Keyword(s):  
Domoic Acid ◽  
Recombinant Antibody ◽  
Variable Region ◽  
Antibody Fragments ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Single Chain Fv ◽  
Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

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