کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5371488 1503955 2010 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimisation of a multivalent Strep tag for protein detection
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی تئوریک و عملی
پیش نمایش صفحه اول مقاله
Optimisation of a multivalent Strep tag for protein detection
چکیده انگلیسی

The Strep tag is a peptide sequence that is able to mimic biotin's ability to bind to streptavidin. Sequences of Strep tags from 0 to 5 have been appended to the N-terminus of a model protein, the Stefin A Quadruple Mutant (SQM) peptide aptamer scaffold, and the recombinant fusion proteins expressed. The affinities of the proteins for streptavidin have been assessed as a function of the number of tags inserted using a variety of labelled and label-free bioanalytical and surface based methods (Western blots, microarray assays and surface plasmon resonance spectroscopy). The binding affinity increases with the number of tags across all assays, reaching nanomolar levels with 5 inserts, an observation assigned to a progressive increase in the probability of a binding interaction occurring. In addition a novel interfacial FRET based assay has been developed for generic Strep tag interactions, which utilises a conventional microarray scanner and bypasses the requirement for expensive lifetime imaging equipment. By labelling both the tagged StrepX-SQM2 and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.

Graphical AbstractResearch Highlights►Multi-Strep tag aptamers show enhanced affinity to streptavidin binding. ►Multi-Strep tags show similar binding properties across a variety of bioanalytical platforms. ►Multi-Strep tags have established the basis for an assay compatible with a future FRET based label-free biosensors.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biophysical Chemistry - Volume 152, Issues 1–3, November 2010, Pages 170-177
نویسندگان
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