nanobiotechnology: an interdisciplinary challenge, 2006

NANOBIOTECHNOLOGYAn Interdisciplinary Challenge

Uwe B. SLEYTRCenter for NanoBiotechnology

University of Natural Resources and Applied Life Sciences Vienna

Center for NanoBiotechnologyUniversity of Natural Resources and Applied Life Sciences Vienna

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

www.biotec.boku.ac.at/znb.html

… exploitation of the results is performed in close collaboration with Nano-S

Research groups:

• Nanoengineering (Margit Sára)

• Nanoglycobiology (Paul Messner)

• Nanostructures (Dietmar Pum)

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

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

mod. after Pielartzik, Bayer

Nanosciences and Nanotechnology

„Self-assembly“

Microlithography

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 ofBranched 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

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.

Description of S-layers

For review see: Sleytr et al., 1999, Angew. Chemie Int. Ed., 38:1034-1054

S-layer Lattice Typesoblique square

hexagonal

p3 p6

p1 p2 p4

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

a 10 nm b 10 nmc 10 nm

Ultrastructure of S-layer Protein Lattices

Three dimensional image reconstruction of an S-layer with square (p4) lattice symmetry

AFM image of an S-layerwith square (p4) latticesymmetry

AFM image of an S-layerwith oblique (p2) latticesymmetry

Properties of S-layer Lattices

Patterning elements for a molecular construction kit

S-layer lattices are composedof identical species of subunits.

Pores passing through showidentical size and morphology.

Functional groups are alignedin 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 MembranesExploiting Isoporosity

S-layer

Microfiltration membraneas support

S-layer Ultrafiltration Membranes

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

Sharp Cut Off

Conventional Functionalizationof Surfaces

Functionalization of S-layer Lattices(Binding of Molecules and Nanoparticles on S-layers)

Repetitive physicochemical properties

Pores in S-layers have identical sizeand morphology

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.

• Chemical methods• Design and expression of recombinant S-layer fusion proteins

Examples:

a b c100nm 100nm 200nm

Exact Orientation of Molecules and Functions

Native S-layer protein

S-layer fusion protein

Chemical or genetical functionalization

S-layer + Function (Antibody,

Streptavidin

10 nm

10 nm.

Enzyme , Antigen, Ligand)

Applications of S-layer Fusion ProteinsDiagnostic 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. anti-allergic 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

Silver binding peptideCobalt binding peptide

12 aa12 aa

rSbpA31-1068 / AG4 and AGP35rSbpA31-1068 / CO2P2

Heavy chain camel antibody117 aarSbpA31-1068 / cAb

Green fluorescent protein238 aarSbpA31-1068 / GFP

IgG-Binding domain116 aarSbpA31-1068 / ZZ

Affinity tag for streptavidin9 aarSbpA31-1068 / Strep-tag

Major birch pollen allergen116 aarSbpA31-1068 / Bet v1

Biotin binding118 aarSbsB1-889 / core streptavidinrSbpA31-1068 / core streptavidin

FunctionalityLength of funct.

S-layer fusion protein(selected from various constructs)

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

IgG

Microbead with bound SCWP

ZZ-domains

rSbpA31-1068

Primary circuit (blood)

Secondary circuit(plasma + microspheres)

Microspheres Based Detoxification-System

Cooperation with D. Falkenhagen, V. Weber et al.

Biomimetic Cell Membranes

TEM of an archaeal cell

http://sun.menloschool.org/~cw

eaver

Cell membrane: fluid mosaic model

Specific membrane functions

S-layer

Plasma-membrane1.selectivity 2.binding 3.opening / closing

Archaeal cell envelope structure

A supramolecular structure opti-mized in ~ 3,5 billions of years under extreme environmental conditions (120°C, pH 0, concen-trated salt solutions, 1100 bar).

S-layer Stabilized Solid Supported Lipid Membranes

S-layer

Functionalizedlipid membrane

S-layer

Solid support(e.g. Si-wafer, gold)

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

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

Application Potential ofS-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

bound functional molecules

S-layer protein

S-layerfusion

proteins transmembranefunction

Biomimetic VirusesS-layer Liposomes and Emulsomes

100nm

Potential Applications

• Artificial viruses (inclusion of nucleic acid) for gene therapy.

• Drug-targeting and drug-delivery.

• Vaccines and immune therapy.

• Transport of hydrophobic substances.

EM-Photograph of an S-layer coatedliposome.

HIV VirusSource: WellcomePhoto Library, Medical Art Service, München

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

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

S-layer Coated Lipid / Plasmid Particels

Cell patterningCellular lithography

Backgrounds of cell repulsive material

Grid patterns of cell friendly material

M. Scholl, et al, J. Neurosci. Meth. (2000) 104, 65-75

Summary

• 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).

Nano Sciences and Nano(bio)technology require: