Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor
Autor: | M. Verstraete, Anne Catherine Vanhove, Robert G. Shatters, Kato Deckers, Pierre Rougé, Charles A. Powell, Dov Borovsky |
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Rok vydání: | 2021 |
Předmět: |
ATPase
mosquito E. coli fluorescence microscopy Microbiology Biochemistry law.invention 03 medical and health sciences Affinity chromatography law Complementary DNA medicine Binding site Receptor kinetic characterization Molecular Biology ABC-TMOF receptor 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology molecular modeling 030306 microbiology Chemistry sequencing Trypsin QR1-502 Amino acid Recombinant DNA biology.protein medicine.drug |
Zdroj: | Biomolecules, Vol 11, Iss 934, p 934 (2021) Biomolecules Volume 11 Issue 7 |
ISSN: | 2218-273X |
Popis: | Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-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 Ae. aegypti. The mRNA of the receptor 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 (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli 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 E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide. |
Databáze: | OpenAIRE |
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