Extracellular expression of the HT1 neurotoxin from the Australian paralysis tick in two Saccharomyces cerevisiae strains.

Abstract

Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, and single-chain variable IgG fragments, however, the use of SDL for the expression and purification of proteins is gaining interest. Prokaryote SDL express proteins within the periplasm, limiting the application of common screening techniques, such as ELISA and FACS, to assess the viability of recombinant toxin before purification. A previous attempt to express a functional holocyclotoxin-1 (HT1) from the Australian paralysis tick (Ixodes holocyclus) using a prokaryotic system was unsuccessful. In this study, the coding sequence (CDS) of HT1 was cloned into the pYD1 plasmid and transformed by electroporation into IMTV014 and EBY100 yeast cell lines. Post induction, recombinant HT1 was identified on the cell surface of IMTV014/ht1 and EBY100/ht1 transformants by FACS, Western blot, and ELISA utilizing dog anti-paralysis tick IgG. The recombinant HT1 was purified, and functionality confirmed by an in vitro synaptosome-binding assay. This research reports for the first time the extracellular expression and display of a functional HT1 on the surface of the S. cerevisiae. It also provides evidence that yeast display libraries provide a viable technology to produce recombinant toxins, and their screening using high throughput methodologies such as FACS.

• Authors: Thomas Karbanowicz, Eric Dover, Xinyi Mu, Ala Tabor, Manuel Rodriguez-Valle
• Journal: Toxicon : official journal of the International Society on Toxinology (Toxicon) Vol. 140 Pg. 1-10 (Dec 15 2017) ISSN: 1879-3150 [Electronic] England
• PMID: 29042312 (Publication Type: Journal Article)
• Copyright: Copyright © 2017 Elsevier Ltd. All rights reserved.

Chemical References

• Arthropod Venoms
• Immunoglobulin G
• Neurotoxins
• Recombinant Proteins
• Toxins, Biological
• holocyclotoxin

Topics

• Animals
• Arthropod Venoms (genetics, metabolism)
• Base Sequence
• Cell Surface Display Techniques (methods)
• Dogs
• Immunoglobulin G
• Ixodes (genetics)
• Neurotoxins (genetics, metabolism)
• Recombinant Proteins
• Saccharomyces cerevisiae (genetics, metabolism)
• Toxins, Biological (genetics, metabolism)