smart protein nano-materials with genetically encoded ... · properties of protein nanofiber...
TRANSCRIPT
Smart protein nano-materials with
genetically encoded functionality
Mats Sandgren
Associate Professor
Department of Molecular Sciences, SLU
Nature has already invented
Many existing materials in Nature are composed of
protein nanofibersSuspending fibers for lacewing eggs
Barnacle cement
(Havstulpaner)
Spider web
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)
Many proteins spontaneously form nanofibers
Soluble protein Protein nanofiber
Illustration by Veronica Lendel
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
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
Technology platform for functional nanofibers
Fusion protein
(fiber forming+enzyme)
Fiber forming
protein
Illustration by Veronica Lendel
Functional nanofiber
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)
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
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
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
A protein-fiber based bioreactor
Illustration by Veronica Lendel
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
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
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)
