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

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.

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

PLoS ONE ◽

10.1371/journal.pone.0241773 ◽

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3343-3349.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3343-3349 ◽

Cited By ~ 40

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

BioTechniques ◽

10.2144/btn-2021-0093 ◽

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.

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