A Streptavidin Mutant Useful for Directed Immobilization on Solid Surfaces

Abstract

A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.

Sensing Immobilized Molecules of Streptavidin on a Silicon Surface by Maldi-Tof Mass Spectrometry and Fluorescence Microscopy

“…The streptavidin/biotin system is of special interest because it has one of the largest free energies of association yet observed for the non-covalent binding of a protein and small ligand in aqueous solution (K a = 10 13 M -1 ) 8 . Streptavidin is a highly stable protein that can maintain its functional structure even under a variety of harsh conditions, such as high temperatures, etc 9 . These unique properties, along with the ability of biotin to be easily incorporated into various biological materials without disturbing their properties and structures, allow streptavidin to serve as a powerful, versatile tool in biomedical science and clinical diagnostics.…”

“…One group of streptavidin applications involves solid phase assays, in which streptavidin is immobilized on a solid surface 9,11 . The extremely high biotin-binding affinity of streptavidin allows tight, efficient immobilization or capture of biotinylated materials on streptavidin-coated solid surfaces.…”

A robust method for production of MHC tetramers with small molecule fluorophores

“…Expression and purification of StvC was as described by Reznik et al (Reznik et al, 2001), with the following modifications. DTT was included throughout the entire purification procedure at a concentration of 5 mM.…”

“…Following published protocols, we expressed StvC subunits as inclusion bodies in E. coli, washed them with a mild detergent, solubilized them in a denaturing buffer, and refolded them by dialysis (Reznik et al, 2001). We replaced the published purification on an iminobiotin affinity matrix with purification on a Phenyl-sepharose hydrophobic interaction column, permitting elution of the protein under milder conditions.…”

“…Though we have tried-and failed-to reproduce this procedure in our own laboratory, the original hypothesis led us to consider methods for targeted labeling of streptavidin at a site remote from the biotin-binding site. While wild-type streptavidin does not have a good candidate site such as a free cysteine thiol, the groups of Cantor and Sano have developed an E. coli expression system for recombinant streptavidin (Sano and Cantor, 1990), including a mutant with a single cysteine residue at the carboxylterminus of each of the four identical subunits that they refer to as StvC (Reznik et al, 2001). We now report the robust production of high-quality MHC tetramers using based upon StvC labeled with maleimide derivatives of a variety of fluorophores.…”