Abstract
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 rSbpA31-1178/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 rSbpA31-1178/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.
| Original language | English |
|---|---|
| Pages (from-to) | 895-903 |
| Number of pages | 9 |
| Journal | Bioconjugate Chemistry |
| Volume | 20 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 20 2009 |
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ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Organic Chemistry
- Pharmaceutical Science
- Biomedical Engineering
- Pharmacology
- Medicine(all)
Cite this
Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays. / Badelt-Lichtblau, Helga; Kainz, Birgit; Völlenkle, Christine; Egelseer, Eva Maria; Sleytr, Uwe B.; Pum, Dietmar; Ilk, Nicola.
In: Bioconjugate Chemistry, Vol. 20, No. 5, 20.05.2009, p. 895-903.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays
AU - Badelt-Lichtblau, Helga
AU - Kainz, Birgit
AU - Völlenkle, Christine
AU - Egelseer, Eva Maria
AU - Sleytr, Uwe B.
AU - Pum, Dietmar
AU - Ilk, Nicola
PY - 2009/5/20
Y1 - 2009/5/20
N2 - 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 rSbpA31-1178/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 rSbpA31-1178/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.
AB - 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 rSbpA31-1178/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 rSbpA31-1178/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.
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U2 - 10.1021/bc800445r
DO - 10.1021/bc800445r
M3 - Article
VL - 20
SP - 895
EP - 903
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 5
ER -