A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery

Abstract

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Venom biotechnology: casting light on nature’s deadliest weapons using synthetic biology

“…The snake venom kallikrein was later expressed in the same system, and the bioactivity was similar to its natural counterpart in vitro ( Wang et al, 2012 ). Other cell-free production systems have recently been compared for their ability to produce an ICK toxin from the sicariid spider Hexophthalma dolichocephala ( Lüddecke et al, 2021 ). This showed that only one system (based on E. coli ) was able to produce a bioactive toxin in low amounts and unclear folding, whereas the other systems failed to yield any product at all ( Lüddecke et al, 2021 ).…”

“…Other cell-free production systems have recently been compared for their ability to produce an ICK toxin from the sicariid spider Hexophthalma dolichocephala ( Lüddecke et al, 2021 ). This showed that only one system (based on E. coli ) was able to produce a bioactive toxin in low amounts and unclear folding, whereas the other systems failed to yield any product at all ( Lüddecke et al, 2021 ). The authors therefore recommended the optimization of cell-free systems and protocols to maximize protein yield and folding specificity ( Lüddecke et al, 2021 ).…”

“…This showed that only one system (based on E. coli ) was able to produce a bioactive toxin in low amounts and unclear folding, whereas the other systems failed to yield any product at all ( Lüddecke et al, 2021 ). The authors therefore recommended the optimization of cell-free systems and protocols to maximize protein yield and folding specificity ( Lüddecke et al, 2021 ). The most recent application of cell-free systems for venom toxin production involved the modification of a thermodynamically controlled E. coli -based cell-free system (AC-assisted Ec CFS) to produce a variety of complex, bioactive folded peptides from a range of sources.…”

Heilen mit Tiergiften

“…Hierfür steht mittlerweile eine ganze Bandbreite an Methoden zur Verfügung, wobei die hetero loge Expression in Bakterienzellen noch immer am häufigs ten eingesetzt wird. In jüngerer Vergangenheit wurden aber auch Technologien in eukaryotischen Zellen und so gar vollkomen zellfreie Ansätze verwendet um Bestandteile aus Tiergiften im Labor herzustellen [17][18][19].…”

Enlighting the toxinological dark matter of spider venom enzymes

“…More recent venomics strategies combine proteomics and transcriptomics to shed light on the composition of venoms [21]. When combined with biotechnology, this approach provides access to all venom proteins and peptides in a given source [22]. A major insight from modern venomics is that spider venoms not only contain small ICK peptides but also an array of high-molecular-weight proteins, often with putative enzymatic activity [2,23].…”