Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata

Abstract

Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C–C–CC–C–C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides.

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins

“…Recombinant synthesis allows for high yield and purity, but does not easily permit the incorporation of unnatural amino acids or site-specific labeling of peptides. The typically small volume of venom produced by terebrids requires multiple synthetic approaches including both chemical and recombinant synthesis methods [ 72 , 111 ].…”

“…More recently substituting select cysteines for selenocysteines [ 113 , 118 ] significantly advanced the synthesis and folding of cysteine rich venom peptides. SPPS has been the method of choice for the synthesis of several conotoxins and also for incorporation of unnatural amino acids such as d -amino acids [ 43 , 64 , 110 , 111 , 112 , 113 ]. We have recently applied SPPS to successfully synthesize Tv1, a 23-amino acid teretoxin from Terebra variegata [ 72 ] ( Figure 5 B,C).…”

“…We recently described a method for the recombinant expression and characterization of terebrid teretoxin peptide Tgu6.1, from Terebra guttata [ 111 ]. The teretoxin Tgu6.1 is a novel 44-amino acid teretoxin peptide with a cysteine scaffold similar to the VI/VII framework (C-C-CC-C-C) of the I, M, and O-superfamilies found in cone snails.…”

“…In the case of Tgu6.1, thioredoxin was introduced in the plasmid for disulfide folding and solubility issues, His6-tag and Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography were used as a purification method, and enterokinase was applied to site-specifically cleavage Tgu6.1 from the fusion protein. The recombinantly expressed Tgu6.1 peptide exhibited bioactivity, displaying a paralytic effect when tested in a bioassay using the native prey or terebrids, Nereis virens (Annelida) [ 111 ].…”

“…Teretoxin polychaete assays are conducted by injecting the folded terebrid peptide in the ventral nerve cord of a polychaete. Two additional polychaetes are usually injected with saline solution as a negative control, and a well-characterized peptide toxin (e.g., agatoxin) as a positive control [ 39 , 72 , 111 ]. Two recently characterized teretoxins, Tv1 and Tgu6.1, analyzed using this bioassay caused partial paralysis in Nereis virens polychaetes [ 72 , 111 ].…”