In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date the biochemical pathways through which IAG exerts its effects is poorly understood and could be elucidated using the production of a recombinant (r) IAG. The objective of the present study was to express glycosylated, properly folded, and biologically active IAG using the Pichia pastoris yeast expression system. Using recombinant proteins, we aimed to develop specific tools to control sex determination by administration of IAG at the critical sexual differentiation time window. The methylotrophic yeast P. pastoris was used to co-express recombinant single-chain precursor molecules consisting of the B and A chain (the mature hormone) tethered by a flexible linker. We produced rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). Western blot analysis of the recombinant proteins, resolved by SDS-PAGE, yielded bands of ~10 kDa for rSv-IAG, ~11 kDa for rCq-IAG and rMr-IAG as their expected molecular sizes. When tested for their biological activity and ability to enhance phosphorylation in the testis, the rIAGs significantly and in a dose-dependent manner enhanced phosphorylation specific to their species and some managed to elicit response in other species as well. Moreover we identified, using Bioinformatics analysis of the de-novo assembled spiny lobster transcriptome, the spiny lobster insulin receptor. We further validated this discovery with a receptor activation assay in Cos-7 cells expressing the receptor with a reporter SRE-LUC system designed for RTKs, using recombinant IAG proteins of three decapods species. This study might further enhance the development of new methods to facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological role of IAGs in crustaceans.
2015 University Research Conference: Integrate, Innovate, Inspire, Sunshine Coast, Australia 13-16 July 2015