Animal venoms constitute a library of bioactive, disulfide-rich peptides and proteins that exhibit extraordinary pharmacological properties with established potential as research tools and therapeutics. This is illustrated by predatory marine cone snails (Conus) which use complex venoms for prey capture and defense. The venoms of the ~800 species of cone snails represent a particularly rich source of toxins (conotoxins) with diverse molecular targets, such as ion channels, receptors and transporters. However, recombinant expression of conotoxins in E. coli is hindered, especially due to the complexities of forming the disulfide bonds, critical for folding and maintaining structural integrity. In this work, we identify the sequence of a previously uncharacterized conotoxin, Mu8.1 from Conus mucronatus, that defines a new conotoxin gene superfamily. Using the established csCyDisCo bacterial expression system, we successfully produce Mu8.1 in E coli. We determine the crystal structure of Mu8.1 and find that it has structural homology with conotoxins of the con-ikot-ikot superfamily and that both Mu8.1 and con-ikot-ikot display a saposin-like fold. Functional testing demonstrates that Mu8.1 selectively blocks calcium flux in a subset of neurons, the peptidergic nociceptors, known to convey painful stimuli to the central nervous system. We show that Mu8.1 blocks voltage-gated calcium channels, most potently Cav2.3 (R-type calcium channels), when tested against a range of recombinant ion channels. We substantiate the Cav2.3 inhibition by showing that calcium flux in Mu8.1-responsive cells is also blocked by SNX-482, a known toxin inhibitor of Cav2.3. These results raise the possibility that Mu8.1 could be a new molecular probe for Cav2.3 channels and for identification of functional subclasses of peptidergic nociceptors. We have also identified a new conotoxin, CTX3 from Conus geographus, that belongs to a documented but uncharacterized gene superfamily. We express CTX3 using csCyDisCo and preliminary investigations show a fully oxidized protein. The successful expression of Mu8.1 and CTX3 demonstrate the feasibility of detailed structural and functional investigations of larger conotoxins which have largely been neglected due to difficulties in producing them. Finally, we contribute to the molecular toolkit for bacterial expression of disulfide-rich proteins by the design of two DisCoTune (Disulfide bond formation in E. coli with tunable expression) plasmids. We show that by combining the tunable expression capabilities from the pLEMO vector with the foldases from (cs)CyDisCo, we increase the stability of these two expression systems thus promoting higher yields of correctly folded protein. The two systems are showcased with the expression of an industrially relevant proteinase, Proteinase K, and a conotoxin, Conk-S3.