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 non-
life 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 packag-
ing of organic or biomaterial devices
or assemblies in which the dimen-
sion of at least one functional comp-
onent lies between 1 and 100nm.

• Nanobiotechnology is
characterized by its highly inter-
disciplinary nature and features a
close collaboration between life-
scientists, 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?

ƒ Biological molecules (e.g. proteins, lipids, glycans,

nucleic acids)
ƒ Chemically or genetically modified molecules
ƒ Chemically synthesized molecules
 Key Capabilities

ƒ Supramolecular design
ƒ Self-assembly strategies and morphogenesis
(sequential assembly routes)
ƒ Patterning elements
• Monomolecular arrays
• Vesicles
• Tubes
ƒ Combination of "top-down“ and "bottom-up“ strategies
ƒ Functionalization of supramolecular structures
ƒ 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 AFM image of an S-layer AFM image of an S-layer

reconstruction of an S- with square (p4) lattice with oblique (p2) lattice
layer with square (p4) symmetry symmetry
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, 10 nm

Enzyme , Antigen, Ligand)

Native S-layer protein

Streptavidin

10 nm.

S-layer fusion protein

 Applications of S-layer Fusion Proteins
ƒ Diagnostic systems and label free detections systems
(SPR, SAW, QCM-D)
ƒ High density affinity coatings (e.g. biocatalysis, blood
purification (MDS))
ƒ Immunogenic and immunomodulating structures (e.g. anti-
allergic vaccines)
ƒ Stabilization of functional lipid membranes
ƒ Functionalization of liposomes and emulsomes as targeting and
delivery systems
ƒ Biomineralization
ƒ Binding of nanoparticles (e.g. molecular electronics, non linear
optics)
 S-layer Fusion Proteins
S-layer fusion protein Length Functionality
(selected from various constructs) of funct.
rSbsB1-889 / core streptavidin 118 aa Biotin binding
rSbpA31-1068 / core streptavidin
rSbpA31-1068 / Bet v1 116 aa Major birch pollen allergen

rSbpA31-1068 / Strep-tag 9 aa Affinity tag for streptavidin

rSbpA31-1068 / ZZ 116 aa IgG-Binding domain

rSbpA31-1068 / GFP 238 aa Green fluorescent protein

rSbpA31-1068 / cAb 117 aa Heavy chain camel antibody

rSbpA31-1068 / AG4 and AGP35 12 aa Silver binding peptide

rSbpA31-1068 / CO2P2 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 opti-
http://sun.menloschool.org/~cweaver

mized in ~ 3,5 billions of years

under extreme environmental
conditions (120°C, pH 0, concen-
trated salt solutions, 1100 bar).

Cell membrane: fluid mosaic model

Specific membrane functions

Plasma-
1.selectivity 2.binding 3.opening / closing membrane

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 Schuster et al., 1998, Biochim. Biophys. Acta Biomembr.1370:280-288.
Schuster et al., 2001 Langmuir 17: 499-503
• tethering structures Schuster et al., 2002, Bioelectrochem. 55:5-7
• ionic reservoir 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.
• Drug-targeting and drug-delivery.
EM-Photograph of
an S-layer coated • Vaccines and immune therapy.
liposome.
100nm • Transport of hydrophobic substances.
 S-layer Coated Lipid / Plasmid Particels

Electron micrograph of ultrathin section demon- Photograph of HeLa cells expressing green fluor-
strating the internalization of S-layer coated lipid / escent protein after transfection with S-layer
plasmid particles into HeLa cells. 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).