Dataset Information

Streptavidin Coverage on Biotinylated Surfaces.

ABSTRACT: Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the functionalization density is required to tune the platform's properties. Streptavidin (SAv) monolayers are widely used to immobilize biotinylated proteins, receptors, and DNA. The SAv density on a surface can be varied easily, but the predictability is dependent on the method by which the SAv is immobilized. In this study we show a method to quantitatively predict the SAv coverage on biotinylated surfaces. The method is validated by measuring the SAv coverage on supported lipid bilayers with a range of biotin contents and two different main phase lipids and by using quartz crystal microbalance and localized surface plasmon resonance. We explore a predictive model of the biotin-dependent SAv coverage without any fit parameters. Model and data allow to predict the SAv coverage based on the biotin coverage, in both the low- and high-density regimes. This is of special importance in applications with multivalent binding where control over surface receptor density is required, but a direct measurement is not possible.

SUBMITTER: Hamming PHE

PROVIDER: S-EPMC8662640 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Streptavidin Coverage on Biotinylated Surfaces.

Hamming P H Erik PHE Huskens Jurriaan J

ACS applied materials & interfaces 20211123 48

Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the functionalization density is required to tune the platform's properties. Streptavidin (SAv) monolayers are widely used to immobilize biotinylated proteins, receptors, and DNA. The SAv density on a surface can be varied easily, but the predictability is dependent on the method by which the SAv is immobilized. In this study we sho ...[more]

PMID: 34813287

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Project description:Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Dataset Information

Streptavidin Coverage on Biotinylated Surfaces.

Publications

Streptavidin Coverage on Biotinylated Surfaces.

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