NANOBIOTECHNOLOGY An Interdisciplinary Challenge

Uwe B. SLEYTR
Center for NanoBiotechnology University of Natural Resources and Applied Life Sciences Vienna

Center for NanoBiotechnology
University of Natural Resources and Applied Life Sciences Vienna

Head: Uwe B. Sleytr
Development of a supramolecular construction kit based on monomolecular crystalline protein lattices (S-layers) as patterning elements for life and nonlife science applications.

Research groups: • • • Nanoengineering (Margit Sára)

Nanoglycobiology (Paul Messner) Nanostructures (Dietmar Pum)

… exploitation of the results is performed in close collaboration with Nano-S
www.biotec.boku.ac.at/znb.html

Definition of NanoBiotechnology
• Nanobiotechnology involves the
processing, fabrication and packaging of organic or biomaterial devices or assemblies in which the dimension of at least one functional component lies between 1 and 100nm.

• Nanobiotechnology is
characterized by its highly interdisciplinary nature and features a close collaboration between lifescientists, physical scientists, and engineers.

courtesy of FEI-Company, NL

Converging Technologies

mod. after Pielartzik, Bayer

NT is not a „rebranding“ of older science ! Its influence is revolutionary rather than evolutionary.

Nanosciences and Nanotechnology
Microlithography

„Self-assembly“

Which are the Basic Building Blocks in a Biomolecular Construction Kit?

Biological molecules (e.g. proteins, lipids, glycans, nucleic acids) Chemically or genetically modified molecules Chemically synthesized molecules

Key Capabilities
Supramolecular design Self-assembly strategies and morphogenesis (sequential assembly routes) Patterning elements • Monomolecular arrays • Vesicles • Tubes Combination of "top-down“ and "bottom-up“ strategies Functionalization of supramolecular structures Characterization

Basic Structures (Patterning Elements) for Generating Complex Supramolecular Structures

• DNA • Monomolecular crystalline bacterial cell surface layers
(S-layers)

Nanoscale Assembly and Manipulation of Branched DNA (Ned Seeman, NY University)

http://seemanlab4.chem.nyu.edu/homepage.html

Bacterial Surface Layer Proteins (S-layers)

AFM image of an S-layer with square (p4) lattice symmetry (d=13.1nm) reassembled on a silicon wafer.
Center for NanoBiotechnology and BOKU, Vienna, Austria

Description of S-layers
S-layers are crystalline, monomolecular (glyco)protein arrays representing one of the most commonly observed surface structures in eubacteria and archaea.

TEM of freeze etched preparations of bacterial cells with an S-layer showing square (left) and hexagonal (right) lattice symmetry. For review see: Sleytr et al., 1999, Angew. Chemie Int. Ed., 38:1034-1054

S-layer Lattice Types
oblique square

p1

p2

p4

hexagonal

p3

p6

Center-to-center spacing of the morphological units in the range of 3 – 30 nm.

Ultrastructure of S-layer Protein Lattices

a

10 nm

b

10 nm

c

10 nm

Three dimensional image reconstruction of an Slayer with square (p4) lattice symmetry

AFM image of an S-layer with square (p4) lattice symmetry

AFM image of an S-layer with oblique (p2) lattice symmetry

Properties of S-layer Lattices
Patterning elements for a molecular construction kit
S-layer lattices are composed of identical species of subunits. Pores passing through show identical size and morphology. Functional groups are aligned in well defined positions and show defined orientations.

Nano(bio)technological Applications of S-layers

• Isoporosity • Sterically defined functionality • Coating of Surfaces • Components for supramolecular structures
(molecular LEGO)

S-layer Ultrafiltration Membranes
Exploiting Isoporosity

S-layer

Microfiltration membrane as support

S-layer Ultrafiltration Membranes
Sharp Cut Off

Myoglobin (Mr 17 000) Carbonic anhydrase (Mr 30 000) Ovalbumin (Mr 43 000) Bovine serum albumin (Mr 67 000)

Conventional Functionalization of Surfaces

Functionalization of S-layer Lattices
(Binding of Molecules and Nanoparticles on S-layers)
• • Chemical methods Design and expression of recombinant S-layer fusion proteins

Pores in S-layers have identical size and morphology

Repetitive physicochemical properties

Functional domains (native or genetically introduced) are repeated with the periodicity of the S-layer lattice leading to regular arrays of bound molecules and particles.

Examples:

a

100nm

b

100nm

c

200nm

Exact Orientation of Molecules and Functions
Chemical or genetical functionalization

S-layer

+ Function (Antibody,

Enzyme , Antigen, Ligand)

10 nm

Native S-layer protein Streptavidin

10 nm.

S-layer fusion protein

Applications of S-layer Fusion Proteins
Diagnostic systems and label free detections systems (SPR, SAW, QCM-D) High density affinity coatings (e.g. biocatalysis, blood purification (MDS)) Immunogenic and immunomodulating structures (e.g. antiallergic vaccines) Stabilization of functional lipid membranes Functionalization of liposomes and emulsomes as targeting and delivery systems Biomineralization Binding of nanoparticles (e.g. molecular electronics, non linear optics)

S-layer Fusion Proteins
S-layer fusion protein
(selected from various constructs)

Length of funct.

Functionality

rSbsB1-889 / core streptavidin rSbpA31-1068 / core streptavidin rSbpA31-1068 / Bet v1 rSbpA31-1068 / Strep-tag rSbpA31-1068 / ZZ rSbpA31-1068 / GFP rSbpA31-1068 / cAb rSbpA31-1068 / AG4 and AGP35 rSbpA31-1068 / CO2P2

118 aa Biotin binding 116 aa Major birch pollen allergen 9 aa Affinity tag for streptavidin 116 aa IgG-Binding domain 238 aa Green fluorescent protein 117 aa Heavy chain camel antibody 12 aa Silver binding peptide 12 aa Cobalt binding peptide

Mature proteins: Bacillus sphaericus CCM2177 variant A (SbpA): 1238 aa Geobacillus stearothermophilus PV72/p2 (SbsB): 889 aa

Microspheres Based Detoxification-System
Secondary circuit (plasma + microspheres)

IgG ZZ-domains rSbpA31-1068

Primary circuit (blood)
Cooperation with D. Falkenhagen, V. Weber et al.

Microbead with bound SCWP

Biomimetic Cell Membranes
Archaeal cell envelope structure A supramolecular structure optimized in ~ 3,5 billions of years under extreme environmental conditions (120°C, pH 0, concentrated salt solutions, 1100 bar).
Cell membrane: fluid mosaic model
http://sun.menloschool.org/~cweaver

Specific membrane functions
1.selectivity 2.binding 3.opening / closing
Plasmamembrane

TEM of an archaeal cell

S-layer

S-layer Stabilized Solid Supported Lipid Membranes
S-layer Functionalized lipid membrane S-layer Solid support (e.g. Si-wafer, gold)

S-layer proteins as: • stabilizing structures • tethering structures • ionic reservoir

Schuster et al., 1998, Biochim. Biophys. Acta Biomembr.1370:280-288. Schuster et al., 2001 Langmuir 17: 499-503 Schuster et al., 2002, Bioelectrochem. 55:5-7 Schuster et al., 2003, Langmuir 19:2392-2397

Application Potential of S-layer Supported Lipid Membranes

• Exploiting functional lipid membranes at meso- and
macroscopic scale: ~30 % of all proteins found in various organisms are membrane proteins > 50 % of the proteins interact with membranes ~ 15 % of the most sold drugs act on ion channels ~ 60 % of the ethical drugs affect membrane proteins

• Linking silicon technology and solid state physics with
biological systems (e.g. coupling cells to surfaces) Biosensors, HTScreening, Diagnostics, Lab-on-a-chip

Biomimetic Viruses
S-layer Liposomes and Emulsomes
S-layer protein S-layer fusion proteins

transmembrane function

HIV Virus
Source: Wellcome Photo Library, Medical Art Service, München

bound functional molecules

Potential Applications • Artificial viruses (inclusion of nucleic acid) for gene therapy.
EM-Photograph of an S-layer coated liposome.
100nm

• Drug-targeting and drug-delivery. • Vaccines and immune therapy. • Transport of hydrophobic substances.

S-layer Coated Lipid / Plasmid Particels

Electron micrograph of ultrathin section demonstrating the internalization of S-layer coated lipid / plasmid particles into HeLa cells.

Photograph of HeLa cells expressing green fluorescent protein after transfection with S-layer coated lipid/ pEGFP-C1 particles

Cell patterning
Cellular lithography
Backgrounds of cell repulsive material

Grid patterns of cell friendly material
M. Scholl, et al, J. Neurosci. Meth. (2000) 104, 65-75

Summary Nano Sciences and Nano(bio)technology require:

• Transdisciplinary strategies and integration of methods • • •
of Life- and Non Life Sciences as part of Converging Technologies. Optimal size (teams, equipment, infrastructure....). The BOKU has set priorities. Long-term development strategies (national and international grants). Adapted tertiary education: PhD-program involving different universities (Converging Universities).

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