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

Toxins

卷:13

André Billion¹  Tim Lüddecke¹  Kim N. Kirchhoff¹  Andreas Vilcinskas¹  Anne Paas¹  Thomas Timm²  Günter Lochnit²  Björn M. von Reumont³  Lea Talmann⁴ 

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

[2] Institute of Biochemistry, Justus Liebig University of Giessen, Friedrichstr. 24, 35392 Giessen, Germany;

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

[4] Syngenta Crop Protection, Werk Stein, Schaffhauserstrasse, CH4332 Stein, Switzerland;

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

DOI  :  10.3390/toxins13080575

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown