Cloning and Characterization of Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor.

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female and cross linked to His-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of . The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. 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 = 113.7 ± 18 nM ± SEM and B = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.