کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2021227 | 1069236 | 2009 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain
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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
The expressed recombinant protein contains an N-terminal (His)6-tag, a hemagglutinin(HA)-tag, and an 11-amino acid HIV-derived TAT-membrane transduction domain and was purified to homogeneity by Sephacryl S-100 molecular exclusion and Ni2+-affinity chromatography. The purified TAT-LMP-1 protein was chemically labeled with fluorescein, and its time and concentration dependent entry into rabbit blood cells was monitored by flow cytometry. We demonstrate the accumulation of TAT-tagged LMP-1 both in cytoplasmic and nuclear compartments. By performing affinity pull-down assays, we confirm our earlier findings that the recombinant TAT-LMP-1, when used as molecular bait to identify the intracellular binding proteins, interacts with Smurf1, a known binding partner of LMP-1. We also show potentiation of BMP-2 activity using the purified TAT-LMP-1 in mouse muscle C2C12 cells by assaying a heterologous luciferase-reporter construct containing multiple copies of a BMP-responsive sequence motif. Finally, we also confirm the biological activity of the purified TAT-LMP-1 by showing enhancement of BMP-2 induced increase of alkaline phosphatase mRNA and protein by RT-PCR and enzyme activity, respectively.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 65, Issue 2, June 2009, Pages 165-173
Journal: Protein Expression and Purification - Volume 65, Issue 2, June 2009, Pages 165-173
نویسندگان
Sreedhara Sangadala, Motohiro Okada, Yunshan Liu, Manjula Viggeswarapu, Louisa Titus, Scott D. Boden,