کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843157 | 1069185 | 2013 | 13 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Large scale expression and purification of mouse melanopsin-L in the baculovirus expression system
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Melanopsin is the mammalian photopigment that primarily mediates non-visual photoregulated physiology. So far, this photopigment is poorly characterized with respect to structure and function. Here, we report large-scale production and purification of the intact long isoform of mouse melanopsin (melanopsin-L) using the baculovirus/insect cell expression system. Exploiting the baculoviral GP67 signal peptide, we obtained expression levels that varied between 10-30Â pmol/106Â cells, equivalent to 2-5Â mg/L. This could be further enhanced using DMSO as a chemical chaperone. LC-MS analysis confirmed that full-length melanopsin-L was expressed and demonstrated that the majority of the expressed protein was N-glycosylated at Asn30 and Asn34. Other posttranslational modifications were not yet detected. Purification was achieved exploiting a C-terminal deca-histag, realizing a purification factor of several hundred-fold. The final recovery of purified melanopsin-L averaged 2.5% of the starting material. This was mainly due to low extraction yields, probably since most of the protein was present as the apoprotein. The spectral data we obtained agree with an absorbance maximum in the 460-500Â nm wavelength region and a significant red-shift upon illumination. This is the first report on expression and purification of full length melanopsin-L at a scale that can easily be further amplified.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 91, Issue 2, October 2013, Pages 134-146
Journal: Protein Expression and Purification - Volume 91, Issue 2, October 2013, Pages 134-146
نویسندگان
Nazhat Shirzad-Wasei, Jenny van Oostrum, Petra H. Bovee-Geurts, Maud Wasserman, Giel J. Bosman, Willem J. DeGrip,