Optimisation of a multivalent Strep tag for protein detection.
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-SQM(2) and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.
Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3QR, United Kingdom.
Michael
M
Busby
Lukas Kurt Josef
LK
Stadler
Paul
P
Ko Ferrigno
Jason J
JJ
Davis
D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000737:chemistry; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D055312:Cystatin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000345:Affinity Labels; C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D052158:Aptamers, Peptide; D055312:Cystatin A; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin