A fluorescent screening method for optimization of conotoxin expression in Pichia pastoris.

Lei Zheng, Zeyin Lin, Haiping Fan, Mengxue Chen, Jiawei Yu, Ying Miao, Binghua Wu

Biotechnology and applied biochemistry 2022 Aug

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Conotoxins are small cysteine-rich peptides secreted by the Conus venom glands, which act on ion channels or membrane receptors with high specificity and potency. Conotoxins are invaluable sources for neuroscience research and drug leads, but their application is hindered by the limited successes in quantitative engineering using either chemical or biotechnological approaches. Here, we explore the Pichia pastoris to express 23 selected conopeptides using a GFP-based fluorescence screen. We found that, in a protease-deficient strain PichiaPink™ Strain 4 (ade2 prb1 pep4), most of the recombinant conopeptides were expressed as two major folding variants including a compact form that was somehow resistant to reduction and high temperature. The GFP-αTxIA was the only one displaying a single band that showed a dose-dependent neurotoxicity on larvae of the insect Plutella xylostella, with a 48-h LD50 lower than 1.12 pmol mg-1 body weight. Furthermore, the recombinant αTxIA after cleavage from the fusion was able to inhibit cell proliferation of the LYCT and HEK293T cell lines with an appearance IC50 of 341 ± 8 and 235 ± 15 nM, respectively. This screening method is straightforward and easy to scale up, providing a versatile tool for further optimization of conotoxin production in the yeast cell. © 2021 International Union of Biochemistry and Molecular Biology, Inc.

Citation

Lei Zheng, Zeyin Lin, Haiping Fan, Mengxue Chen, Jiawei Yu, Ying Miao, Binghua Wu. A fluorescent screening method for optimization of conotoxin expression in Pichia pastoris. Biotechnology and applied biochemistry. 2022 Aug;69(4):1611-1621