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

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Conotoxins, derived from Conus venom, are potent modulators of ion channels and receptors.
• Their application in neuroscience and drug discovery is limited by challenges in quantitative engineering.
• Efficient biotechnological production of conotoxins remains a significant hurdle.

Purpose of the Study:

• To develop and validate a high-throughput screening method for expressing and identifying functional conopeptides in Pichia pastoris.
• To assess the neurotoxic and antiproliferative activities of engineered conotoxins.
• To optimize conotoxin production using a protease-deficient yeast strain.

Main Methods:

• Utilized Pichia pastoris, a protease-deficient strain (PichiaPink™ Strain 4), for recombinant conopeptide expression.
• Employed a GFP-based fluorescence screen to identify successfully expressed and folded conopeptides.
• Assessed neurotoxicity using Plutella xylostella larvae and antiproliferative effects on LYCT and HEK293T cell lines.

Main Results:

• Successfully expressed 23 selected conopeptides, with most appearing as two folding variants.
• Identified GFP-αTxIA as a single-band variant exhibiting potent, dose-dependent neurotoxicity in Plutella xylostella (48-h LD50 < 1.12 pmol mg-1).
• Recombinant αTxIA demonstrated significant inhibition of LYCT and HEK293T cell proliferation (IC50 values of 341 ± 8 nM and 235 ± 15 nM, respectively).

Conclusions:

• Developed a scalable and straightforward GFP-based screening method for conopeptide discovery and production in yeast.
• Demonstrated the potential of Pichia pastoris for engineering bioactive conotoxins, exemplified by the potent αTxIA.
• The findings provide a versatile platform for optimizing conotoxin yields and exploring their therapeutic potential.