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

journal contribution
submitted on 2024-06-27, 09:40 and posted on 2024-06-27, 10:08 authored by Vijay Gupta, Indulekha P. Sudhakaran, Zeyaul Islam, Nishant N. Vaikath, Issam Hmila, Tamas Lukacsovich, Prasanna R. Kolatkar, Omar M. A. El-Agnaf

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.

Other Information

Published in: PLOS ONE
License: http://creativecommons.org/licenses/by/4.0/
See article on publisher's website: https://dx.doi.org/10.1371/journal.pone.0241773

Funding

Hamad Bin Khalifa University, Qatar Biomedical Research Institute (SF 2017-007)

History

Language

  • English

Publisher

Public Library of Science (PLoS)

Publication Year

  • 2020

License statement

This Item is licensed under the Creative Commons Attribution 4.0 International License.

Institution affiliated with

  • Hamad Bin Khalifa University
  • Qatar Biomedical Research Institute - HBKU
  • Diabetes Research Center - QBRI
  • Neurological Disorders Research Center - QBRI