Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

Abstract

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

Identification of a Flavonoid Glucosyltransferase Involved in 7-OH Site Glycosylation in Tea plants (Camellia sinensis)

“…The molecular interaction docking analysis of CsUGT75L12 was performed with UDP-Glc as the sugar donor and flavonol (kaempferol) as the acceptor, using Molegro virtual docker software 35 . The result of docking data were evaluated using lowest energy values.…”

Sensitive and Specific Protein Sensing Using Single-Mode Tapered Fiber Immobilized With Biorecognition Molecules

“…Their strong non-covalent protein-ligand interaction, with a high dissociation constant value of K D ¼ 10 À15 M [10], has made them very useful in applications concerning drug delivery [11], labeling [12], and detection [13]. By having one part, either biotin or Avidin, bound on the surface of the sensor, any biomolecules can be easily immobilized provided that they are labeled with the complement.…”

The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

“…The tetrameric bacterial protein streptavidin (SAV) is one of the most commonly used protein reagents in biotechnology because of its intrinsically high level of binding specificity and affinity (Green, 1990;Wilchek & Bayer, 1990;Laitinen et al, 2007;O'Sullivan et al, 2012). In addition to the cognate ligand biotin, SAV binds strongly to a number of peptide ligands.…”