Proteins for Nano-technology

Application of fermentation to other filed

Sugarcane

Sugar factory

Harvest

Syrup and Biomass

Fermentation factory

Bi-product: Fertilizer

Functional Bio-product (Protein)

Renewable devices

Recycling

CO2

Protein character #1：Structure 1

0 n

m

Bio-molecules made self-assemble into functional structures.

Cage-shaped

Tube

Motor

Flower

David S. Goodsell and the RCSB PDB

By pya!

Bio-polymer (Conchiolin) Argonite crystal CaCO3

By Micrographia HP

20 nm

Diatom cell wall

Ca

Si

NPs by protein in blood

Fe

Protein character #2：Inorganic affinity

Bio-molecules can recognize various inorganic materials.

Core Bio-Material; Cage-shaped protein, ferritin and Dps

12

nm

9 n

m

David S. Goodsell and the RCSB PDB

Metal ion

Protein mutants for the Bio Nano Process

We can design proteins to applications by genetic engineering.

Placement on Si-layer ⇒ + Si-binding peptides

Placement on CNT ⇒ + CNT-binding peptides

Deposition of TiO2-layer ⇒ + Ti-mineralizing peptides

etc…

・Metal (Ti, Si)-binding ・TiO2, SiO2-mineralization ・NPs formation

Sequence of TBF (181 a.a., M. W. = 20.6kDa) MRKLPDASSQIRQNYSTEVEAAVNRLVNLYLRASYTYLSLGFYFDRDDVALEGVCHFFRELAEEKREGAERLLKMQNQRGGRALFQDLQKPSQDEWGTTPDAMKAAIVLEKSLNQALLDLHALGSAQADPHLCDFLESHFLDEEVKLIKKMGDHLTNIQRLVGSQAGLGEYLFERLTLKHD

Metal-binding cage-shaped protein Ti-Binding-Ferritin (TBF)

minTBP-1:

Metal-binding site

Horse ferritin (24 mer)

K. Sano (2005) Small. 1, 826

Sequence of CDT1 (177 a.a., M. W. = 20.5kDa) MDYFSSPYYEQLFMKTINSVDTKEFLNHQVANLNVFTVKIHQIHWYMRGHNFFTLHEKMDDLYSEFGEQMDEVAERLLAIGGSPFSTLKEFLENASVEEAPYTKPKTMDQLMEDLVGTLELLRDEYKQGIELTDKEGDDVTNDMLIAFKASIDKHIWMFKAFLGKAPLEMRKLPDA

Bi-functional cage-shaped protein CNHBP-Dps-TBP(CDT1)

NHBP-1:

CNT-binding site

minTBP-1:

Ti-mineralization site

L. innocua Dps (12 mer)

WO2012/086647 I. Inoue (2011) Chem. Commun. 47, 12649

・CNT, Metal(Ti, Si)-binding ・TiO2, SiO2-mineralization ・NPs formation

pET20-protein

Protein gene

TBF and CDT1 protein was produced by E. coli

fermentation. 20nm

12nm

Production of Functional Proteins

20nm

9nm

TBF (Ferritin variant)

CDT1 (Dps variant)

Purification of CDT1

40

30

20

15

CDT1 (20.5kDa)

kDa M 1 2

M : Marker 1 : BL21(DE3)/pET20b 2 : BL21(DE3)/pET20-CDT1

0

500

1000

1500

0 100 200 300 400 500 600

Ab

s28

0n

m(u

nit

) Gel filtration

Elution volume (ml)

0

200

400

600

800

1000

1200 NaC

l con

centratio

n (m

M)

0

50

100

150

200

0 200 400 600 800

Elution volume (ml)

Ab

s28

0n

m(u

nit

)

Abs280 NaCl conc.

Ion exchange

20 nm

20 nm

TBF (Ferritin variant)

CDT1 (Dps variant) + Iron ion at 4℃ for 3 – 4 hours

the RCSB PDB

TBF

CDT1

CNT-binding activity by QCM

0

1.6

CDT1 w/o CNT-BP

Ka

(x 1

08, M

)

1.4

1.2

1.0

0.8

0.6

0.4

0.2

The CNT-binding activity is improved by 50-times.

CNT sensor

Protein

Time Time CDT1 Dps

Titanium(IV) bis-(ammonium lactato)-

dihydroxide CDT1

Absorbance at 660nm

Incubate at r. t.

pH6.0

n = 7

Ti-mineralizing activity of CDT1

0

0.1

0.2

0.3

0.4

0 2 4 6 8

Ti-

min

era

lizati

on

(u

nit

s)

Time (h)

Dps-minTBP1

Dps

water

The Ti-mineralizing activity is improved by minTBP1.

50 nm

Fe-NPs

Grow In C2H2/H2 H2 activation

FeOx cored cage-shaped protein

CNTs-growth; NPs as catalysts

CNT coated with CDT1 Biomineralization of TiO2-layer

CDT1s

CNT

50 nm

Construction of CNT-TiO2 nanocomposite

CNT

TiO2 film

50 nm

CoMoCAT CNT (SG65) with CDT1

The CDT1s were able to bind the semi-conductive SWNTs