Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays.
The mesophilic organism Lysinibacillus sphaericus CCM 2177 produces the surface (S)-layer protein SbpA, which after secretion completely covers the cell surface with a crystalline array exhibiting square lattice symmetry. Because of its excellent in vitro recrystallization properties on solid supports, SbpA represents a suitable candidate for genetically engineering to create a versatile self-assembly system for the development of a molecular construction kit for nanobiotechnological applications. The first goal of this study was to investigate the surface location of 3 different C-terminal amino acid positions within the S-layer lattice formed by SbpA. Therefore, three derivatives of SbpA were constructed, in which 90, 173, or 200 C-terminal amino acids were deleted, and the sequence encoding the short affinity tag Strep-tag II as well as a single cysteine residue were fused to their C-terminal end. Recrystallization studies of the rSbpA/STII/Cys fusion proteins indicated that C-terminal truncation and functionalization of the S-layer protein did not interfere with the self-assembly capability. Fluorescent labeling demonstrated that the orientation of the crystalline rSbpA(31-1178)/STII/Cys lattice on solid supports was the same, like the orientation of wild-type S-layer protein SbpA on the bacterial cell. In soluble and recrystallized rSbpA/STII/Cys fusion proteins, Strep-tag II was used for prescreening of the surface accessibility, whereas the thiol group of the end-standing cysteine residue was exploited for site-directed chemical linkage of differently sized preactivated macromolecules via heterobifunctional cross-linkers. Finally, functionalized two-dimensional S-layer lattices formed by rSbpA(31-1178)/STII/Cys exhibiting highly accessible cysteine residues in a well-defined arrangement on the surface were utilized for the template-assisted patterning of gold nanoparticles.
Badelt-Lichtblau
Helga
H
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Kainz
Birgit
B
Völlenkle
Christine
C
Egelseer
Eva-Maria
EM
Sleytr
Uwe B
UB
Pum
Dietmar
D
Ilk
Nicola
N
D000818:Animals; D001406:Bacillaceae / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D003545:Cysteine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D053768:Metal Nanoparticles / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013499:Surface Properties; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C120455:SbpA protein, bacteria; D014867:Water; D006046:Gold; D002256:Carbonic Anhydrases; D003545:Cysteine