Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays

Helga Badelt-Lichtblau, Birgit Kainz, Christine Völlenkle, Eva Maria Egelseer, Uwe B. Sleytr, Dietmar Pum, Nicola Ilk

Research output: Contribution to journalArticlepeer-review

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 languageEnglish
Pages (from-to)895-903
Number of pages9
JournalBioconjugate Chemistry
Volume20
Issue number5
DOIs
Publication statusPublished - May 20 2009

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Organic Chemistry
  • Pharmaceutical Science
  • Biomedical Engineering
  • Pharmacology
  • Medicine(all)

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