Production and activity evaluation of Heliothis virescens recombinant prothoracicotropic hormone

The prothoracicotropic hormone (PTTH) is a neuropeptide that, interacting with the TORSO receptor, stimulate prothoracic glands (PGs), triggering a cascade of events that lead to the synthesis of ecdysone, the molting hormone. It is produced in neurosecretory cells of the brain together with other hormones (insulin-like factor and prothoracicostatic hormone, the natural PTTH antagonist). PTTH is synthesized as pre-pro-hormone and subsequently processed in the brain, to obtain a glycosylated homodimer with active subunits of 109 amino acids, with intra- and extra-chain disulfide bonds. The study of ecdysteroidogenesis by in vitro incubation of PGs is generally performed using a crude brain extract. This method does not allow to standardize the dose and amount of PTTH to be used in incubation tests, since the brain homogenate represents a complex matrix of components with unknown relative proportions which make the results of such experiments highly variable. In this work, the recombinant active form of PTTH (rPTTH) of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) has been produced. The main goal is to obtain a purified rPTTH suitable to perform in vitro tests, in a standardized and repeatable manner. Avoiding the use of the crude brain extract would allow determining the real dose of rPTTH which is required to synthesize and secrete ecdysone. We evaluated the functionality and stimulatory effect of rPTTH by in vivo and in vitro tests on H. virescens last instar larvae PGs.