Cloning and characterization of Aedes aegypti trypsin-modulating oostatic factor (TMOF) gut-receptor

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to (His6)-TMOF.  The crosslinked complex was purified by Ni affinity chromatography. SDS PAGE identified 2 protein bands (45 kDa and 61 kDa).  The  bands were cut digested and  analyzed using MS/MS identifying a protein sequence (1307 amino acids) in the genome of Ae. aegypti. The mRNA of the gene was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA^- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell.  The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (K_D = 113.7 + 18 nM + SEM and B_max = 28.7 + 1.8 pmol + SEM).  Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor not only binds TMOF but also imports it into the bacterial cells indicating that the receptor’s role is to import the hormone into the mosquito’s gut epithelial cells. The receptor was named ABC-TMOF-importer.  A 3D modeling of the receptor indicates that the receptor has ATP binding sites at the N and C-termini of the receptor.  Incubation of the recombinant E. coli cells with Na Arsenate and Na Azide, ATPase inhibitors,  significantly reduced the import of TMOF-FITC into the bacterial cells.   Valinomycin and DNP  did not inhibit TMOF transport, indicating that TMOF gut receptor is an ABC-importer.