title: Recombinant expression of Drosophila melanogaster α-L-fucosidase in Trichoplusia ni cells.
abstract: 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 80kDa. 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.
authors.affiliation: Institute of Insect Science, Zhejiang University, Hangzhou, China.
authors.forename: Yipeng
authors.initials: Y
authors.lastname: Xu
authors.forename: Jari
authors.initials: J
authors.lastname: Intra
authors.forename: Chuan-xi
authors.initials: CX
authors.lastname: Zhang
authors.forename: Maria Enrica
authors.initials: ME
authors.lastname: Pasini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity; D029721:Drosophila Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D005260:Female; D005854:Germ Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D005644:alpha-L-Fucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005644:alpha-L-Fucosidase