کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5922273 | 1165362 | 2011 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Recombinant expression of Drosophila melanogaster α-l-fucosidase in Trichoplusia ni cells Recombinant expression of Drosophila melanogaster α-l-fucosidase in Trichoplusia ni cells](/preview/png/5922273.png)
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.
Highlights⺠We expressed the Drosophila melanogaster α-l-fucosidase in Trichoplusia ni cells. ⺠The recombinant protein contains the six histidine motif. ⺠The purified protein interacts with α-l-fucose residues on the micropyle. ⺠The α-l-fucosidase is involved in mechanisms of gamete interactions in fruit fly.
Journal: Journal of Insect Physiology - Volume 57, Issue 9, September 2011, Pages 1205-1211