Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

Abstract

Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.(Strept)avidin with reversible biotin binding capability can extend the applications of the biotin-(strept)avidin technology. These molecules can be applied for affinity purification of biotinylated biomolecules, screening of ultratight binders binding to biotinylated biomolecules displayed on the phage display system, and development of reusable biosensor chips, protein/ antibody microarrays, and enzyme bioreactors (1, 2). (Strept)avidin is a homotetrameric molecule with a biotin binding site in each subunit (3). The three-dimensional structure of (strept)avidin (4, 5) suggests that a complete biotin binding pocket in each subunit requires the contribution of a tryptophan residue from an adjacent subunit. Site-directed mutagenesis studies also demonstrate the importance of this residue for tight biotin binding and subunit communications (6 -8). Therefore, development of monomeric (strept)avidin can be an attractive approach to engineer (strept)avidin with reversible biotin binding capability.The engineering of (strept)avidin to its monomeric form is technically challenging. In the case of avidin, the first generation of engineered monomeric avidin can exist in the monomeric state only in the absence of biotin (9). This problem has been solved by the recent development of the second generation of monomeric avidin (10), which carries two mut...

Genetically engineered avidins and streptavidins

“…These monomeric streptavidins, based on interface residue modifications, showed approximately four times weaker affinity toward biotin when compared with monomeric streptavidin, where monomerization was achieved by mutating biotin-binding residues T90A and D128A [54,55,67]. In the same study Wu and Wong [67] also constructed a streptavidin mutant where they mimicked our strategy to monomerize avidin. A double mutant (Q95A, W120K) (analogous to the monoavidin containing mutations N54A and W110K) was, however, oligomeric but showed a strong biotin-binding affinity collapse, similar to our W120K streptavidin mutant [65].…”

“…They reported the K d of the resultant mutant to be 1.3 × 10 -8 M toward biotin and showed successful utilization of the immobilized mutant in the purification of biotinylated cytochrome C from a bacterial extract, and then its mild elution by desthiobiotin. In a more recent study, the same group used a rational approach to change several amino acid residues in all subunit interfaces to generate electrostatic repulsion and steric hindrance between subunits [67]. Mutations of Val55, Thr76 and Val125 residues into either arginine or threonine were utilized.…”

“…In the initial construct, the ATD sequence of streptavidin (amino acids [65][66][67] in the loop between β-strands four and five was converted to RGD by site-directed mutagenesis. This construct did not, however, promote cell adhesion in an in vitro experiment with rat aortic endothelial and human melanoma cells, possibly due to inadequate exposure or a sterically unfavorable structure of the domain.…”

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles

“…Besides the natural glycoprotein avidin (pI ∼ 10), analogue proteins expressed in bacteria or recombinant proteins without carbohydrates and a near-neutral pI are available, the most common being streptavidin and neutravidin. Also monomeric streptavidin with, however, reduced affinity to biotin has been reported (Wu & Wong 2005). By genetic modification resulting in deactivated binding pockets, a tetrameric but monovalent streptavidin derivative with only one single binding site for biotin has also been recently demonstrated .…”

Quantum dots–DNA bioconjugates: synthesis to applications

“…Each streptavidin has four biotin binding sites, thereby enabling association of more than one DNA per protein. In order to further control the stoichiometry of biotin on streptavidin, molecularly engineered variants of the protein have been used, which have affinity for only one biotin [74,75] (and hence biotinylated DNA) that permit monolabelling ( per streptavidin molecule) with biotinylated rsfs.royalsocietypublishing.org Interface Focus 6: 20160064 DNA [76]. Molecular cloning has also realized facile production of higher affinity avidin variants such as dimeric rhizavidin [77,78] or electrostatically neutral variants (at physiological pH) such as neutravidin which further expands the tool-kit of this type of conjugation.…”