A cDNA encoding an α-l-fucosidase from Drosophila melanogaster was obtained from the recombinant plasmid named pGEM-DmFuca and inserted into the pBacHTeGFPT vector to construct the recombinant donor plasmid which was transposed to the target AcBacmid in Escherichia coli (DH10) by Tn7 transposition function. The AcBacmid-GFP-DmFuca plasmid was used to transfect Tn-5B1-4 cells of the Cabbage looper Trichoplusia ni. SDS-PAGE analysis revealed a band of about 80. kDa. Using a polyclonal antiserum raised against α-l-fucosidase protein from D. melanogaster Western blotting analysis confirmed that the fusion protein eGFP-DmFuca has been successfully expressed in a biologically active form in Tn-5B1-4 cells. The recombinant protein, containing the histidine-tag motif, was purified using an affinity chromatography column. In vitro binding assays the purified eGFP-DmFuca interacts with α-l-fucose residues present on the micropyle of the D. melanogaster eggshell, confirming that the α-l-fucosidase is a good candidate as receptor involved in gamete interactions in fruit fly.
Recombinant expression of Drosophila melanogaster α-l-fucosidase in Trichoplusia ni cells / Y. Xu, J. Intra, C.X. Zhang, M.E. Pasini. - In: JOURNAL OF INSECT PHYSIOLOGY. - ISSN 0022-1910. - 57:9(2011), pp. 1205-1211.
|Titolo:||Recombinant expression of Drosophila melanogaster α-l-fucosidase in Trichoplusia ni cells|
INTRA, JARI (Secondo)
PASINI, MARIA ENRICA (Ultimo)
|Parole Chiave:||Baculovirus system; Fertilization; Fruit fly; Fucose; Green fluorescent protein|
|Settore Scientifico Disciplinare:||Settore BIO/06 - Anatomia Comparata e Citologia|
|Data di pubblicazione:||2011|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.jinsphys.2011.05.008|
|Appare nelle tipologie:||01 - Articolo su periodico|