In vivo and in vitro surface display of heterologous proteins on Bacillus thuringiensis vegetative cells and spores

The Bacillus surface display system is a heat-stable platform for heterologous protein expression that preserves biological activity in an adverse environment. In the present study, a new cell surface display system that enables heterologous proteins to be displayed consecutively on the surface of Bacillus thuringiensis vegetative cells and spores in vivo and in vitro is reported. Using the N-terminal domain (Mbgn) of a peptidoglycan hydrolase (Mbg) as anchor, the green fluorescence protein (GFP) and a bacterial laccase (WlacD) can be targeted onto the surface of vegetative cells and spores in vivo in an active form. This was confirmed by GFP fluorescence intensity quantification, enzymatic activity measurement, immunofluorescence microscopy examination, and flow cytometry assay. Immobilization of externally added Mbgn-mediated fusion proteins in vitro to obtain actively displaying GFP and/or WlacD was subsequently achieved in B. thuringiensis, Bacillus subtilis and Bacillus cereus, demonstrating the versatility of the proposed system. Therefore, Mbgn-mediated in vivo and in vitro immobilization of foreign proteins represents an improved method of microbial display for extended biotechnological applications.