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

Author

Vijay Gupta, Indulekha P Sudhakaran, Zeyaul Islam, Nishant N Vaikath, Issam Hmila, Tamas Lukacsovich, Prasanna R Kolatkar, and Omar M A El-Agnaf

Subjects
Medicine, Science
Abstract

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.

Published
2020
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2. Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies

Author

Zeyaul Islam, Omar M. A. El-Agnaf, Tamas Lukacsovich, Indulekha P. Sudhakaran, Issam Hmila, Nishant N. Vaikath, Prasanna R. Kolatkar, and Vijay Gupta

Subjects
Models, Molecular, 0301 basic medicine, Protein Denaturation, Protein Folding, Protein Expression, Protein Engineering, Biochemistry, Inclusion bodies, law.invention, 0302 clinical medicine, Protein structure, law, Macromolecular Structure Analysis, Urea, Enzyme-Linked Immunoassays, Cloning, Molecular, Inclusion Bodies, Multidisciplinary, Organic Compounds, Chemistry, Monomers, Molecular Docking Simulation, Denaturation, Physical Sciences, alpha-Synuclein, Recombinant DNA, Medicine, Protein folding, Research Article, Protein Binding, Protein Structure, Science, Research and Analysis Methods, 03 medical and health sciences, Gene Expression and Vector Techniques, Escherichia coli, Humans, Homology modeling, Molecular Biology Techniques, Immunoassays, Molecular Biology, Imidazole, Synucleinopathies, Molecular Biology Assays and Analysis Techniques, Binding Sites, Organic Chemistry, Chemical Compounds, Biology and Life Sciences, Proteins, Protein engineering, Polymer Chemistry, 030104 developmental biology, Docking (molecular), Immunologic Techniques, 030217 neurology & neurosurgery, Single-Chain Antibodies
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.

Published
2020

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