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 journalArticle

22 Citations (Scopus)

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

Fingerprint

Genetic engineering
Trp-Ser- His-Pro-Gln-Phe-Glu-Lys
Genetic Engineering
Proteins
Cysteine
Crystal lattices
Self assembly
Amino acids
Amino Acids
Fusion reactions
Crystalline materials
Protein C
Sulfhydryl Compounds
Gold
Nanoparticles
Crystal symmetry
Macromolecules
Crystal orientation
Labeling
S-layer proteins

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 journalArticle

Badelt-Lichtblau, Helga ; Kainz, Birgit ; Völlenkle, Christine ; Egelseer, Eva Maria ; Sleytr, Uwe B. ; Pum, Dietmar ; Ilk, Nicola. / Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays. In: Bioconjugate Chemistry. 2009 ; Vol. 20, No. 5. pp. 895-903.
@article{272558da851546dc94f0cc275fb29114,
title = "Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays",
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.",
author = "Helga Badelt-Lichtblau and Birgit Kainz and Christine V{\"o}llenkle and Egelseer, {Eva Maria} and Sleytr, {Uwe B.} and Dietmar Pum and Nicola Ilk",
year = "2009",
month = "5",
day = "20",
doi = "10.1021/bc800445r",
language = "English",
volume = "20",
pages = "895--903",
journal = "Bioconjugate Chemistry",
issn = "1043-1802",
publisher = "American Chemical Society",
number = "5",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=66149174918&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=66149174918&partnerID=8YFLogxK

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 -