smart protein nano-materials with genetically encoded ... · properties of protein nanofiber...

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)

Contact information:Mats.Sandgren@slu.se

Dept. of Molecular Sciences, SLU, Uppsala

200 nm