Biochemical and Biophysical Research Communications
Identification of human α-synuclein specific single chain antibodies☆
Section snippets
Antibodies
Commercially available antibodies were utilized for synuclein and scFv detection (mouse anti-α-synuclein; BD Biosciences, San Jose, CA; mouse monoclonal anti-M2 Flag™; Sigma–Aldrich, St. Louis, MO). Horseradish peroxidase-conjugated (HRP) secondary antibodies were from GE Healthcare. In some cases primary antibodies were HRP-conjugated and used for direct detection (anti-M2 Flag™-HRP, Sigma; anti-HA-HRP, Roche Diagnostics, Indianapolis, IN).
Bacterial expression and purification of α-synuclein
The bacterial expression vector pRK172 containing
Identification of anti-synuclein single-chain antibodies
In its native form human wild-type α-synuclein (SYN) exists as a random coil but can misfold into oligomers and large molecular weight aggregates. Since misfolded SYN is toxic in both cell culture and animal models, we bacterially produced and biochemically purified human SYN which was subsequently experimentally induced to misfold. We then utilized both monomeric and misfolded SYN to pan against a human phage library harboring single-chain antibodies (scFv). Specifically, monomeric SYN was
Discussion
Since SYN is a likely important contributor to Parkinson’s disease pathogenesis and given that its tendency to misfold may be integral in its toxicity we sought to identify scFvs that recognize both native and misfolded forms of SYN. Here, we describe three classes of scFvs that have specificity for: (1) monomeric and aggregated SYN; (2) monomeric, aggregated, and DA-modified SYN; (3) DA-modified proteins exclusively. Of those scFvs that interact with monomeric SYN we have identified specific
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We thank Dr. Semyon Papernov and the Laboratory for Laser Energetics at the University of Rochester for help with atomic force microscopy and Andrew Tompkins and Karthik Venkatesh for technical assistance. This work was supported by DAMD17-03-1-0009 to H.J.F.