A robust platform for recombinant production of animal venom toxin modulators of ion channels
Abstract
ABSTRACT:
Background and Purpose: Peptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions.
Experimental Approach: 13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca 2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding.
Key Results: Monovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK)…
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