Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein

Abstract

CorrectionsCELL BIOLOGY. For the article ''The NF1 tumor suppressor critically regulates TSC2 and mTOR,''

Genetically engineered avidins and streptavidins

“…Similarly, in the B case the avidin mutant begins with β-strand five and ends after β-strand four [73,75,77]. Moreover, in C the sequence begins with β-strand six and ends after β-strand five [73,75].…”

“…Moreover, in C the sequence begins with β-strand six and ends after β-strand five [73,75]. Finally, in D the streptavidin form begins with β-strand eight and ends after β-strand seven [77]. The biotin-binding site of avidin resides at the same end of the antiparallel β-barrel as the new termini in cases A, C and D, reflecting the biotin-binding affinity of the resultant proteins.…”

“…In addition, scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure that is fully functional with four high-affinity biotin-binding sites. Streptavidin has also been modified to have two binding sites in one polypeptide chain (EMBL: AY884152; [77]). In this case two different circularly permuted forms of streptavidin (Fig.…”

“…6), one beginning with β-strand eight and ending after β-strand seven, was fused to another form beginning with β-strand five and ending after β-strand four. The novel streptavidin form was also further modified by introducing point mutations, and the mutants were expressed employing phage display and panning with biotinylated beads (EMBL: AY884153, EMBL: AY884154; [77]). Surprisingly, it was observed that these novel streptavidin forms displayed high affinity towards biotin-4-fluorescein conjugate (K d = 10 -10 -10 -11 M), whereas the affinity to biotin was significantly reduced in these proteins (K d = 10 -5 -10 -6 M) compared with wt streptavidin.…”

Rapid and sensitive detection of rare cancer cells by the coupling of immunomagnetic nanoparticle separation with ELISA analysis

“…The presented method takes advantage of these characteristics, using the CD3 and CD4 biomarkers to distinguish the T helper cells (targets) from the B cells. The tight binding affinity of biotin-streptavidin [15][16][17] enables linkage to the target T helper cells through biotinylated anti-CD3 antibody and HRP-stv. The membranes of the T helper cells thus conjugated with anti-human CD4-MNPs through the antigenantibody reaction, yielding the MNP-bound T helper cells.…”