pAW series of cipAfragexpression vectors and strategy for complex assembly. (A) Vectors were designed for facilitated insertion of fragments of the gene encoding the cellulosomal scaffold protein CipA, into AscI-NotI restriction sites. Scaffolds can be optionally expressed with or without an N-terminal nuclease reporter and/or a C-terminal cell wall anchor motif. pAW304 is designed for expression, secretion, and cell wall-targeting of CipA fragments (CipAfrags) as fusions with the N-terminal NucA reporter. pAW305 is designed for the expression and secretion of CipAfrags as a fusion with the N-terminal NucA reporter, but without the C-terminal anchor motif. pAW504 is designed for expression, secretion, and cell wall-targeting of CipAfrags without the N-terminal NucA reporter. pAW505 is designed for the expression and secretion of CipAfrags with neither the N-terminal NucA reporter nor the C-terminal anchor motif. (B) Graphic depiction of the surface-display strategy of engineered scaffolds and their association with the β-glucuronidase-dockerin fusion protein (UidA-dock1). All successfully displayed CipAfrags are portrayed as fusions with both NucA and a cell wall anchor, however were also expressed and tested without these two components.
Wieczorek and Martin Microbial Cell Factories 2010 9:69 doi:10.1186/1475-2859-9-69