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

Lüddecke, Tim; Paas, Anne; Talmann, Lea; Kirchhoff, Kim N.; von Reumont, Björn M.; Billion, André; Timm, Thomas; Lochnit, Günter; Vilcinskas, Andreas

doi:10.3390/toxins13080575

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

^1,2,*,†

^1,†,

³,

^2,4

¹,

⁵

⁵

and

Andreas Vilcinskas

^1,2,4

Branch for Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Ohlebergsweg 12, 35392 Giessen, Germany

LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), Senckenberganlage 25, 30325 Frankfurt am Main, Germany

Syngenta Crop Protection, Werk Stein, Schaffhauserstrasse, CH4332 Stein, Switzerland

⁴

Institute for Insect Biotechnology, Justus Liebig University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

⁵

Institute of Biochemistry, Justus Liebig University of Giessen, Friedrichstr. 24, 35392 Giessen, Germany

Author to whom correspondence should be addressed.

^†

These authors contributed equally.

Toxins 2021, 13(8), 575; https://doi.org/10.3390/toxins13080575

Received: 5 July 2021 / Revised: 15 August 2021 / Accepted: 17 August 2021 / Published: 18 August 2021

(This article belongs to the Section Animal Venoms)

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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. View Full-Text

Keywords: neglected venomous arthropods; spider venom; knottin; biotechnology; synthetic biology; cell-free expression

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Lüddecke, T.; Paas, A.; Talmann, L.; Kirchhoff, K.N.; von Reumont, B.M.; Billion, A.; Timm, T.; Lochnit, G.; Vilcinskas, A. A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery. Toxins 2021, 13, 575. https://doi.org/10.3390/toxins13080575

AMA Style

Lüddecke T, Paas A, Talmann L, Kirchhoff KN, von Reumont BM, Billion A, Timm T, Lochnit G, Vilcinskas A. A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery. Toxins. 2021; 13(8):575. https://doi.org/10.3390/toxins13080575

Chicago/Turabian Style

Lüddecke, Tim, Anne Paas, Lea Talmann, Kim N. Kirchhoff, Björn M. von Reumont, André Billion, Thomas Timm, Günter Lochnit, and Andreas Vilcinskas. 2021. "A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery" Toxins 13, no. 8: 575. https://doi.org/10.3390/toxins13080575

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