کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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7126 | 535 | 2011 | 8 صفحه PDF | دانلود رایگان |
We introduce the strategic development of self-assembling peptide/protein fragments based on the far-red fluorescent protein mPlum. The first beta strand (mPlum 1, 18 amino acids) of mPlum was engineered to spontaneously bind with the rest of the protein (mPlum 2–11, next 10 beta strands) and to form a native chromophore. The target beta strand mPlum 1 was separated from mPlum 2–11 and linked via a flexible peptide linker, resulting in fluorescently inactive circularly permuted mPlum protein (CpmPlum). In vitro evolution of this CpmPlum to a fluorescently active form and the subsequent splitting of the engineered mPlum 1 peptide afforded self-assembling mPlum fragments. Recombinantly expressed and synthetically prepared beta strand peptides were successfully assembled with the remaining mPlum protein in vitro and in cells. This developed pair of peptide/protein fragments was effectively used for peptide tag detection of alpha-synuclein in mammalian cells. Sequential expression of self-assembling mPlum fragments offered an entirely genetically encoded sensing system of naturally unfolded alpha-synuclein.
Journal: Biomaterials - Volume 32, Issue 34, December 2011, Pages 9051–9058