Polyelectrolyte multilayer (PE) deposition and S-layer technology have been combined to make novel robust biomimetic surfaces and membranes. Isolated subunits of the bacterial cell surface layer from Bacillus sphaericus CCM2I77 SbpA was self-assembled on PE multilayer supports, with the composition of the multilayer playing a crucial role in determining the structure of the resulting supported protein layers. Flat substrates were studied using atomic force microscopy and neutron reflectometry; protein on suitable PE combinations showed a crystalline structure with lattice constants equal to those found in vivo on bacterial surfaces. The mechanical stability of the S-layer is higher when recrystallized on PEs than directly on silicon supports. The recrystallization process was subsequently used to coat colloidal particles, permitting the determination of zeta potentials before and after coating. Hollow capsules could also be coated in the same way, as proven by various techniques. Our results suggest that electrostatic interactions via divalent cations are important for the assembly process. The results also demonstrate that the versatility of the PE multilayer membranes can be successfully combined with the well-defined surface chemistry and structure of 2D protein crystals.