Direct Heterologous Expression of Cyclic Conotoxin [G22N]Vc1.1 in Yeast

Backbone-cyclized conotoxin Vc1.1 is a potent GABAB receptor agonist and analgesic drug lead for treating neuropathic pain. Here, we describe two yeast-based strategies for the direct recombinant production of this cyclic, disulfide-rich peptide. In the first approach, using Pichia pastoris, a fusion protein comprising the conotoxin precursor and a full-length asparaginyl ligase connected by a poly-His linker was secreted into the culture medium. Cyclization of the secreted α-conotoxin was initiated by a two-step pH adjustment protocol for generating the correctly folded conotoxin. In the second approach, Saccharomyces cerevisiae was engineered to display the activated ligase on its cell surface while secreting Vc1.1 precursors extracellularly. Cyclized peptides were detected in the culture supernatant without further processing. These two strategies provide efficient routes for the direct recombinant production of disulfide-rich and backbone-cyclized peptides.