Immobilization of Bioactive Protein A from Staphylococcus aureus (SpA) on the Surface of Bacillus subtilis Spores

Abstract

Protein A from Staphylococcus aureus (SpA) is a 40-60 kDa cell-wall component, composed of five homologous immunoglobulin (Ig)-binding domains folded into a three-helix bundle. Each of these five domains is able to bind Igs from many different mammalian species. Recombinant SpA is widely used as a component of diagnostic kits for the detection and purification of IgGs from serum or other biological fluids. In this study, purified SpA was adsorbed and covalently linked to Bacillus subtilis spores. Spores are extremely stable cell forms and are considered as an attractive platform to display heterologous proteins. A sample containing about 36 μg of SpA was covalently immobilized on the surface of 4 × 10(10) spores. Spore-bound SpA retained its IgG-binding activity, even after seven consecutive binding and washing steps, suggesting that it can be recycled and utilized several times. FACS analysis revealed that spores with covalently attached SpA had significantly improved fluorescence intensities when compared to those of spores with adsorbed SpA, suggesting that the covalent approach is more efficient than sole adsorption regarding protein attachment to the spore surface.

A comprehensive review on staphylococcal protein A (SpA): Its production and applications

“…In 2014, Gashtasbi et al [70] immobilized the alpha-amylase enzyme on the spore surface by the covalent method using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N hydroxysulfosuccinimide. In contrast to that study in which just one domain of SpA was displayed, in 2015, Ghaedmohammadi et al [53] displayed both full-length and truncated forms of SpA containing 5 IgG-binding domains on the spore surface. In the latter study, SpA was displayed on the surface of B. subtilis spores by two nongenetically approaches including spontaneous adsorption of SpA onto the spore surface, and covalent linkage of this protein to the spore surface proteins.…”

“…In contrast to that study in which just one domain of SpA was displayed, in 2015, Ghaedmohammadi et al [53] displayed both full-length and truncated forms of SpA containing 5 IgG-binding domains on the spore surface. They also suggested that the covalent approach is more efficient than adsorption regarding protein attachment to the spore surface [53,71]. The covalent immobilization method using glutaraldehyde as an efficient and low-cost crosslinker had been developed for the first time to display SpA on the spore surface.…”

“…[ [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] Extracellular expression of recombinant SpA…”

“…Displaying both full length and truncated forms of SpA containing 5 IgG-binding domains on the surface of Bacillus subtilis spores by two nongenetically approaches [53,71] Clinical application of SpA in S. aureus typing (SpA Typing)…”

One-step purification and oriented attachment of protein A on silica and graphene oxide nanoparticles using sortase-mediated immobilization

“…SpA is a virulence factor which is located in S. aureus cell wall and is able to bind Fc immunoglobulins (spatially IgG) of the most of mammalians. 34 Therefore, it can be used for the purication of immunoglobulin or Fc conjugated protein from serum or other proteins. Furthermore, the presence of LPxTG motif in its structure makes it suitable to use as a model protein for the sortase-mediated immobilization on different carriers.…”