LEE, K.J.; KIM, H.W.; MYKLES, D.L.; Colorado State University, Ft. Collins: Cloning of a cDNA encoding molt-inhibiting hormone (MIH) from the land crab Gecarcinus lateralis and expression of recombinant MIH in yeast (Pichia pastoris)

Molt-inhibiting hormone (MIH) is an eyestalk neuropeptide belonging to the crustacean hyperglycemic hormone family. MIH acts at the crustacean molting gland (Y-organ, YO) to inhibit ecdysteroid production and prevent molting. To further investigate YO signaling mechanisms regulating ecdysteroid synthesis, a cDNA encoding MIH of the land crab was cloned from eyestalk neural ganglia. A 1.4kb sequence encoding a 35 aa signal peptide and a 78 aa putative MIH was cloned by a combination of RT-PCR and 3� and 5� RACE. The 78 residue peptide was 62-67% identical to MIH sequences from other crab species. This cDNA was used to express recombinant MIH (rMIH) using a yeast (P. pastoris) expression system. The portion of the cDNA encoding the mature MIH was inserted into the yeast expression vector pPICZ&alpha. This vector contains a yeast-derived alpha factor secretion signal and is designed to direct secretion of recombinant protein into the culture medium. Two constructs were designed to yield either the mature (78aa) rMIH protein or a rMIH fusion protein containing a C-terminal c-myc epitope and polyhistidine tag. DNA sequencing was used to confirm that the reading frame of the final constructs was correct. P. pastoris were transformed with the recombinant vectors and induced with methanol to express the protein. Aliquots of the yeast culture were removed at 24hr intervals over several days. Western blot analysis (using either Callinectes sapidus MIH antiserum or c-myc antiserum) indicated expression and secretion of a rMIH fusion protein into the yeast cell culture media. Purification and testing of biological activity of the rMIH are in progress. Supported by NSF (IBN-9904528).