Surface Display of Recombinant Proteins onBacillus subtilisSpores

Abstract

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescentactivated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 ؋ 10 3 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

GerE-independent expression of cotH leads to CotC accumulation in the mother cell compartment during Bacillus subtilis sporulation

“…Proteins were then electrotransferred to nitrocellulose filters (Bio-Rad) and used for Western blot analysis by standard procedures. The analysis of sporulating cells was performed as previously described (Isticato et al, 2001(Isticato et al, , 2004. Samples were harvested at various times during sporulation and disrupted by sonication in 25 mM Tris (pH 7?5), 0?1 M NaCl, 1 mM EDTA, 15 % (v/v) glycerol and 0?1 mg phenylmethylsulfonyl fluoride ml 21 .…”

Crystal Structure of a Bacterial Endospore Coat Component

“…Despite its importance as a model system for studying the assembly of a multiprotein structure, as a platform for the display of heterologous enzymes or antigens, in pathogenesis and host immune response, and possibly in mediating the germination of spores in the gastrointestinal tract (2,10,11), our knowledge of the molecular mechanisms underlying the assembly of the bacterial spore coat is still scarce. The function of individual coat components is largely unknown, and in only a few cases have the interactions relevant for their assembly been unraveled.…”

Versatile microbial surface-display for environmental remediation and biofuels production

“…Spores can survive indefinitely in a metabolically inactive state, stay intact for millions of years and resist temperatures as high as 90ºC [62]. B. subtilis spores displaying the tetanus toxin fragment C using the spore outer coat protein CotB were used as live vaccines [62,63], as well as for screening of tetrameric streptavidin [64].…”

“…B. subtilis spores displaying the tetanus toxin fragment C using the spore outer coat protein CotB were used as live vaccines [62,63], as well as for screening of tetrameric streptavidin [64].…”