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Smart protein nano-materials with
genetically encoded functionality
Mats Sandgren
Associate Professor
Department of Molecular Sciences, SLU
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Nature has already invented
Many existing materials in Nature are composed of
protein nanofibersSuspending fibers for lacewing eggs
Barnacle cement
(Havstulpaner)
Spider web
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Properties of protein nanofiber biomaterials
Self-assembly (which can be triggered)
Excellent materials properties
Can form films, threads, foams and solids
Biocompatible, biodegradable &
produced in biological processes
Structure and function genetically
encoded (which opens for materials design
by protein engineering)
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Many proteins spontaneously form nanofibers
Soluble protein Protein nanofiber
Illustration by Veronica Lendel
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Nanomaterials compared to micromaterials
One mL material:
10 um beads
Area = 0.6 m2
10 nm wide nanofibers
Area = 400 m2
Total nanofiber length = 13 billion meters
= 300 times the circumference of Earth
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Functionalization of protein nanofibers
Functional fusion protein
Tag for purification Nanofiber-forming peptide Functional protein
Wild type fiber-forming protein
Tag for purification Nanofiber-forming peptide
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Technology platform for functional nanofibers
Fusion protein
(fiber forming+enzyme)
Fiber forming
protein
Illustration by Veronica Lendel
Functional nanofiber
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Fungal prion proteins as basis for
nanofiber engineering
Atomic force microscopy of prion protein
based nanofibers (Benjamin Schmuck)
Spontaneous assembly
Diameter Ø < 10 nm
(human hair Ø ≈ 100,000 nm)
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Examples of material engineering under
genetic control
Fusion protein Applications
IgG-binding domains Antibody capture or purification
Luciferase Biosensor (point-of-care diagnostics)
-lactamase Penicillin degradation
Metal binding Heavy metal capture and removalRare earth metal enrichment
Xylanase Enzymatic fibers – reusability of enzymes
Cytochrome p450 Environmental remediation by oxidative processing (requires electrons)– removal of drugs from waste water
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Examples of needs that can be met
with an antibody-binding nanofiber material
IgG capture and purification - monoclonal antibody (mAb) purification
- diagnostics / blood analysis
Need for more efficient products with higher IgG binding capacity
Point-of-care diagnostics - assess brain damage (S100B levels)
- assess risk of myocardial infarction
(troponin T levels)
Need for high-sensitivity detection in sandwich ELISAs
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An IgG antibody-binding nanofiber
Schmuck et al.. (2017) Biotechnol J 12.
Nanofiber of Sup35N doped with
Sup35N-IgGBD (co-assembly)
IgG (Fc) binding
domains
Properties:
• Subnanomolar microscopic Kd• Holds ca. 1 mg IgG / mg nanofiber
(apr 20x better than Protein A Sepharose)
Applications:
• IgG (mAb) pharmaceuticals production
• Serum analysis/diagnosis
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A protein-fiber based bioreactor
Illustration by Veronica Lendel
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An IgG antibody-binding nanofiber
Schmuck et al.. (2017) Biotechnol J 12.
Nanofiber of Sup35N doped with
Sup35N-IgGBD (co-assembly)
IgG (Fc) binding
domains
1 mL column
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Production of proteins for nano-materials
Yeast– Pichia pastoris (Komagataella pastoris) 1-5 g/L have been reported for heterologously expressed
intra- and extracellular proteins
Filamentous fungus – Trichoderma reesei Industrial strains produce >50 g/L of some heterologously
expressed extracellular proteins
Several other options Saccharomyces, Candida utilis, Aspergillus niger
and various strains of Bacillus
At present: Bacteria – E. coli Production levels of 0.01-0.1 g/L
Pichia pastoris
Trichoderma reesei
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Industrial scale-up
Protein production by yeast
fermentation
(5 to 50 gram protein/liter)
One 10 m3 intermediate fermenter:
50 to 500 kg/batch or 5 to 50 tons/year
Present industrial production of enzymes:
Many 100.000 tons per year (e.g. glucose
isomerase, proteases, laccases)