The functional S-layer protein gene slfB of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 was cloned as a polymerase chain reaction product into the expression vector pET Lic/Ek 30 and heterologously expressed in Escherichia coli Bl21(DE3). The addition of His tags to the N and C termini enabled the purification of the recombinant protein by Ni-chelating chromatography. The Ni binding capacity of the His-tagged recombinant S-layer protein was compared with that of the wild-type S layer. The inductively coupled plasma mass spectrometry analyses demonstrate a significantly enhanced Ni binding capability of the recombinant protein. In addition, the self-assembling properties of the purified modified S-layer proteins were studied by light microscopy and scanning electron microscopy. Whereas the wild-type S-layer proteins re-assembled into regular cylindric structures, the recombinant S-layer proteins reassembled into regular sheets that formed globular agglomerating structures. The nanoporous structure of the protein meshwork, together with its enhanced Ni binding capacity, makes the recombinant S-layer attractive as a novel self-assembling biological template for the fabrication of metal nanoclusters and construction of nanomaterials that are of technical interest.