Technical barriers to molecular farming, a coming practical biotechnology

crunchkingofprussiaBiotechnology

Dec 6, 2012 (4 years and 7 months ago)

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1

Technical barriers to molecular farming,
a coming practical

biotechnology

Technical barriers to molecular farming,
a coming practical

biotechnology

Graduate School of
Gyeongsang

National University

Division of Applied Life Science

Kyun

Oh Lee, Ph. D.

2

Plant biotechnology for the future

1
st

wave 2
nd

wave 3
rd

wave



Agronomic traits

1
st



Plants as factories

3
rd

2
nd



Food q
uality

Value

INGARD

cotton

variety

carrying

Bt

gene

Virus (PRSV) resistant papaya

Foods with enhanced vitamins or iron etc.

Plant
-
made pharmaceuticals (PMPs)

Roundup Ready
turfgrass

3

유전

정보
,

바이오

의약품

치료용

단백질

진단용

단백질

단백질

의약품

시대

호르몬

백신

치료용

항체

Completed

in 2003

Nov. 29, 2007

Personal genome
시대

생명공학

기술

발전에

의한

개인

게놈

분석

서비스



단백질

의약품

시대의

도래


Bio
-
pharmaceutical industry may be worth $200
billion (
2
천억


) by 2015

2008, 2009


top 14 blockbuster
단백질

의약품


시장규모

(
120
조원
)

4

$ 100 billion
=
1,000




=
120




2010


대한민국

국가예산
: 292


8
천억



Rapidly expanding
bio
-
pharmaceutical market


5


유전자

재조합과

세포배양

기술을

통해

생산되는

치료용

단백질인

바이오


약품은

특정한

인체

질병에

대해

특이적으로

작용하기

때문에

전통적

화학합


의약품에

비해

부작용이

적고

효능이

우수함


이러한

이유로

전체

의약품



바이오

의약품에

대한

수요는

지속적으로

증가
하고

있음


삼성경제연구소는

바이오

의약

산업의

시장

규모가

2008


1,080


달러에


2015
년에는

2,000


달러로

급증할

전망

[
바이오

의약

시장

무한팽창



낙오



국가미래

'
시름시름
'(
자료
: 2010.3.11.(

)
한국일

)]

Current production system
for
protein
based

therap
eutics

6

Chinese Hamster Ovar
y
(CHO)

Cells


Not economically feasible for some drugs


Not safe from some pathogen infection



Need to develop new cost efficient substituting
systems

Molecular
f
arming:

a solution to the shortfall

7


Production capacity of plants is easy to scale up


Plants provide
low upstream production costs


Plants are free
from

human pathogens


Plant cells facilitate posttranslational modification of proteins


Plant seeds are easy to store

[



/kg
]

[1



/kg
]


50,000 won/40 kg

Plant Golgi


-
1,2
-
Mannosidase I

N
-
acetyl
-
glucosaminyl
-
transferase I (GnT
-
I)

UDP
-
GlcNAc

Transporter


-
Mannosidase II




Fuc
-
呲ans晥rase

GnT
-


Xyl
-
呲ans晥rase

N
-
ace瑹l
-
Glucosaminidase

Complex 呹pe

F

F

F

X

X

F

Golgi complex


1,2
-
mannosidase I

GnT1

UDP
-
GlcNAc

Transporter


-
mannosidase II

GnT
-


啄U
-
Gal

呲anspor瑥r

GalT

SAT

CMP
-
NeuAc

syn瑨e瑡se

Complex 呹pe glycan

8

단백질

폴딩

제어

식물체

개발



인간화
,

맞춤형

당쇄생산

[
핵심

기반

기술
]

식물

형질전환을

위한

바이오

의약품

유전자의

재조합

식물

형질전환





분화

바이오

의약품

생산

정제

세포배양

재배

Vertical Farm


물을

이용한

바이오

의약품

생산

(
molecular

farming
)


Producing
therapeutic proteins or
industrial products

using plants


Final product is
plant
-
made pharmaceuticals

(PMPs)


Molecular farming
industry may be worth $200 billion(2
천억


) by 2015

9

Pharmaceuticals and industrial products that can
be made using plants

치료용

단백질

진단용

단백질

호르몬

백신

치료용

항체

Invertase

Pharmaceuticals

Industrial products

Plastics,


Oils,

Waxes,


Carotenoids
,


Starches

and

etc
.

Spider silk

Phytase

Trypsin

10

Plants used as bio
-
factories for the production
of pharmaceutical proteins

Alfalfa

Plasma Proteins Foot
-
and
-
mouth disease,
Mannheimia

haemolytica
, Shipping fever (
livestock)

Arabidopsis

Human intrinsic factor (vitamin B12 uptake)

Maize

Anti
-
HIV and Anti herpes Simplex Antibodies
Microbiocides

for pulmonary infection
Mabs

for cancer,
autoimmune disease (rheumatoid arthritis,
Crohns

disease, Vaccines for hepatitis B, Norwalk virus (Travelers
disease), Vaccines and
Mabs

for animal disease prevention
Transmissable

Gastroeneteritis

Vaccine for Pigs
Aprotinin

for blood loss and heart surgery

Lemma

Human tissue
plasminogen

activator for peripheral arterial occlusion, Alpha Interferon

Lettuce

Vaccines for Hepatitis B

Moss
(bryophyte
)

Factor IX for
haemophilia

B

Rice

Alternatives to antibiotics in poultry diets,
Lysozyme

for Gastrointestinal health, Topical infections and
inflammations, B
-
cell lymphoma
idiotype

vaccine

Safflower

Therapeutics and oil
-
body
-
based products for oral and dermal delivery

Spinach

Protective antigen for vaccine against Bacillus

Carrot

Glucocerebrosidase

for
Gaucher

disease

Soybean

Herpes simplex virus 2
-

Tobacco
extensin

signal peptide
-

Anti
-
HSV
-
2 (
IgG
)

Tobacco

Non
-
Hodgkins

B
-
cell lymphoma surface antigen
scFv

TGF
-
b
glucocerebrosidase

for
Gauchers

Syndrome Alpha
galactosidase

for enzyme replacement therapy
IgGs

for prevention of dental decay, common cold, neutralization of
chemotherapeutic drug toxicity GAD 7 cytokines for type 1 Diabetes IL
-
10 for inflammatory bowel disease
Glyocprotein

B from human cytomegalovirus (
hCMV
) Colon cancer surface antigen
-

Murine

IgG

signal peptide


ca扲b穹me

fat
-
sto牡g攠摩do牤敲 捡汬敤l
ca扲b

摩d敡s攠

Tobacco and Corn

Gastric lipase for cystic fibrosis,
Lactoferrin

for gastrointestinal infections and
Sjogran’s

(dry eye) syndrome

Tomato, Potato,
Potato
Tuber,
Banana
(someday!)

Edible vaccines against
Enterotoxigenic

E. coli, Norwalk virus, Hepatitis B,
Vibrio

cholera, Rabies virus
-
intact
Glycoprotein Antimicrobial peptides, Rabbit hemorrhagic disease virus

식물

생산

의약품
(PMP)


임상실험



실용



11

Company

Plants

Cultivation

Products

Disease

State

Protalix

Biotherapeutics

carrot

cell culture

glucocerebrosidase

Gaucher

disease

Phase III trial
Ongoing

Biolex

Therapeutics

duckweed

indoor
chambers

alpha interferon

hepatitis C

Phase II trial Ongoing

SemBioSys

Genetics

safflower

field

insulin

diabetes

Phase I/II trial
Projected late 2008

Meristem

Therapeutics

corn

field

lipase

cystic fibrosis

Phase III trial
Projected late 2008

Ventria

Bioscience

rice

field

lactoferrin
,
lysozyme

Diarrhea

Efficacy trial
Completed

Cobento

Arabidopsis

greenhouse

human intrinsic factor

Vitamin B
-
12
deficiency

Approved In Ukraine

Planet
Biotechnology

tobacco

field

secretory

antibody
vaccine

tooth decay

E.U. approved

Dow
AgroSciences

tobacco

cell culture

poultry vaccine

Newcastle disease

USDA approved

CIGB, Cuba

tobacco

greenhouse

vaccine purification
antibody

hepatitis B

On market

Several “
pharmed
” proteins are close to or on the market as supplements, a vaccine reagent, and a medical device.

[Science 2008; 320, 473
-
475]

Pfizer


Protalix


개발한

당근세포

배양법에

의한

glucocerebrosidase
생산

기술을

선수금

$60 million


후불금

$55 million


구입
, 2010
년에



약을

Taliglucerase alfa
라는

이름으로

출시

계획


[2009


12


뉴욕타임즈
, Nature Biotech. 28, 107
-
108 (2010)

현재

실험

연구용으로

시판되고

있는

식물

생산

바이오

의약품
(PMP)

12

Product


Trade Name

Pharma

Crop

Source of
Genes


Commercial
Purpose

Company
Producing


Company Selling

(product number)

Aprotinin

AproliZean

Corn

Cow


Research and
manufacturing


ProdiGene


NA

Aprotinin

Apronexin



Tobacco

Cow


Research and
manufacturing


Large

Scale

Biology


Sigma (A6103)

Avidin

Recombinant
avidin


Corn


Chicken


Research and
diagnostic reagent


ProdiGene


Sigma (A8706)


B
-
glucuronidase

(GUS)

Not available

Corn


Bacteria


Research and
diagnostic reagent


ProdiGene


NA

Trypsin

TrypZean


Corn


Cow


Research
and

manufacturing


ProdiGene


Sigma (T3568, 3449)


Lactoferrin

Not available


Rice


Human


Research


Ventria

Bioscience


Sigma (L4040)


Lysozyme

Not available


Rice


Human


Research


Ventria

Bioscience


Sigma (L1667)


[Science 2008; 320, 473
-
475]

13

Plant
-
made pharmaceuticals: risks and
concerns


Impact on the environment (GMO)


Food supply contamination; accidental or
intentional gene flow,
cross
-
pollination


Outcrossing

(non
-
target species near field sites
e.g. butterflies, bees, etc)


Allergenicity
,
antigenicity
, immunogenicity


Expression levels


Reproducibility


Different
glycosylation

patterns


Quality and efficacy control of PMPs

14

Technical solutions to the barriers for practical
use of molecular farming


Impact on the environment
:


C
ultivate

in greenhouse
, use vertical
farming facility or cell culture system


Allergenicity
,
antigenicity
, immunogenicity:


Development of plants lacking plant
-
specific sugar residues such as
α
1,3
-
fucose and
β
1,2
-
xylose in the protein
glycan


Different
glycosylation

patterns:


Development of humanized
N
-
glycan

producing plants


Expression levels:


Development of strong tissue specific promoters


Development of t
ranslational enhancer


Use protein
secretory

pathway (signal sequence) and protein storage vacuole


Development of
fusion tags

for efficient protein purification (reduce downstream
processing cost)


Inhibition of gene silencing


Codon

usage optimization


Reproducibility:


Induce callus from homozygous transgenic plants


Quality and efficacy control of PMPs:


Regulation of
unfolded protein response (UPR)

Molecular

farming
기술의

실용화



상용화를

위한

주요

핵심기반기술


15

α
1,6

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

β
1,4

β
1,4

α
2,6

α
2,3

인체

단백질

α
1,3

β
1,4

β
1,4

β
1,2

α
1,6

α
1,3

β
1,2

β
1,2

β
1,3

β
1,3

α
1,4

α
1,4

식물


단백질



N
-
glycosylation


인간화











단백질

고발현



분리

정제

기술

확립








Promoter Affinity tag




단백질

폴딩

증진

기술

개발




















Molecular

farming


핵심기반기술

16

Expression systems for molecular farming

Vegetative systems


Seed
-
based systems


Seed have evolved to naturally store
large amounts of protein


Seed desiccation allows intact
storage for years


Uncouples

harvesting from processing


Allow for an inventory of an intermediate between production and
processing


Easy transfer to cell culture method and protein
secretory

system



Protein (%)

lipid (%)

Carbohydrate (%)

Maize

(
Zea

mays
)

8

4

74

Rice

(
Oryza

sativa)


6 ~ 8

1 ~ 3

75

Barley

(
Hordeum

vulgare
)

12

3

76

Oilseed

rape

(
Brassica

napus
)

22 ~ 24

45 ~ 48

18 ~ 20

Alfalfa

(
Medicago

sativa)

38 ~ 40

8 ~ 9

25 ~ 27

Soybean

(
Glycine

max)

38 ~ 40

18 ~ 20

25 ~ 27

Molecular

farming
기술의

실용화



상용화를

위한

주요

핵심기반기술


17

α
1,6

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

β
1,4

β
1,4

α
2,6

α
2,3

인체

단백질

α
1,3

β
1,4

β
1,4

β
1,2

α
1,6

α
1,3

β
1,2

β
1,2

β
1,3

β
1,3

α
1,4

α
1,4

식물


단백질



N
-
glycosylation


인간화











단백질

고발현



분리

정제

기술

확립








Promoter Affinity tag




단백질

폴딩

증진

기술

개발




















Molecular

farming


핵심기반기술

18

Potential role and effect of the
glycocomponent

of
glycoproteins

Role/effect

Comment

Protein

folding

Glycosylation

can

affect

local

protein

secondary

structure

and

help

direct

folding

of

the

polypeptide

chain
.

Protein

targeting/trafficking

The

glycocomponent

can

participate

in

the

sorting/directing

of

a

protein

to

its

final

destination
.

Ligand

recognition/binding

The

carbohydrate

content

of

antibodies,

for

example,

function

in

antibody

binding

to

monocyte

Fc

receptors

and

interaction

with

complement

component

C
1
q
.

Biological

activity

The

carbohydrate

side

chain

of

gonadotrophins

(specifically

the

-
subunit

N
52

side

chain)

is

essential

to

the

activation

of

gonadotrophin

signal

transduction
.

Stability

Sugar

side

chains

can

potentially

stabilize

a

glycoprotein

in

a

number

of

ways

including

enhancing

its

solubility,

shielding

hydrophobic

patches

on

its

surface,

protecting

from

proteolysis

and

directing

participation

in

intrachain

stabilizing

interactions
.

Regulates

protein

half
-
life

Large

amounts

of

sialic

acid

can

increase

a

glycoprotein's

plasma

half
-
life
.

Exposure

of

galactose

residues

can

decrease

plasma

half
-
life

by

promoting

uptake

through

hepatic

galactose

residues
.

Yeast

N
-
glycosylation

is

of

a

high
-
mannose

type,

driving

rapid

removal

from

circulation

through

mannose

receptors
.

Immunogenicity

Some

glycosylation

motifs

characteristic

of

plant
-
derived

glycoproteins

(often

containing

fucose

and

xylose

residues)

are

highly

immunogenic

in

mammals
.

Protein N
-
glycosylation

in the Endoplasmic

Reticulum (ER) and Golgi Apparatus

19

Lumen

Cytoplasm

Endoplasmic

Reticulum

dolichol

OST

GnT

I

α
-
Man II

GnT

II

β
1,

2
-
xylT

α
1,
3
-
fucT

α
1,
4
-
fucT

β
1,3
-
galT

Golgi Complex

N
-
Acetylglucosamine

Mannose

Fuccose

Glucose

Dolichylphosphate

Protein

Polypeptide chain

Xylose

Galactose

20

N
-
glycosylation

in the ER; Glc
3
Man
9
GlcNAc
2


to Man
8
GlcNAc
2

Glycan

Endoplasmic

Reticulum

Ribosome

mRNA

Oligosaccharyl

Transferase

Calnexin

Folding


-
G汵捯s楤is攠I


-
G汵捯s楤is攠II

E删

-
Ma湮os楤ise

Golgi complex

Glc
3
Man
9
GlcNAc
2

Man
8
GlcNAc
2

: Folded peptide chain

: Glucose

: GlcNAc

: Mannose

: Phosphoric acid

: Nascent polypeptide chain

Dolichol

Man
8
GlcNAc
2

N
-
glycosylation

in
Golgi Apparatus

21

Mammalian Golgi


1,2
-
mannosidase I (ManI)

N
-
acetylglucosaminyltransferase I

(GnTI)

UDP
-
GlcNAc

Transporter


-
m
anno獩da獥s䥉
Ian䥉I


1,6
-
fuco獹ltran獦era獥

N
-
acetylgluco獡minyltran獦era獥 䤠I

(䝮T
-
䥉I

β
1,4
-
g
altosyltransferase (GalT)

Sialyltransferases (SAT)

(Complex type N
-
glycan)

CMP
-
NeuAc

synthetase

UDP
-
Gal

Transporter

: Sialic acid

: Galactose

: Xylose

: Folded peptide chain

: Fucose

: GlcNAc

: Mannose

: Phosphoric acid

Plant Golgi


1,3
-
fucosyltransferase I, II

β
1,2
-
xylosyltransferase

N
-
acetylglucosaminyltransferase I I

(GnT
-
II)

N
-
acetyl

glucosaminidase I, III

(
Paucimannose

type N
-
glycan
)

UDP
-
Gal

Transporter


1,2
-
mannosidase I (ManI)

N
-
acetylglucosaminyltransferase I

(GnTI)

UDP
-
GlcNAc

Transporter


-
m
anno獩da獥s䥉
Ian䥉I

β
1,3
-
g
altosyltransferase (GalT)


1,4
-
fuco獹ltran獦era獥s

Yeast Golgi


1,3
-
Mannosyl
-

transferase


(
MNN1
)


1,6
-
Manno獹l
-

tran獦era獥s(
OCH1
)

Mannan

Type


1,2
-
Mannosyl
-

transferase

(
MNN2,5?
)

Mannose
-
6
-

Phosphate

Synthesis

(
MNN4,6
and others)

n

22

Methods of N
-
glycosylation

humanization

in Plants

GlcNAc

Gal

NeuAC

Man

Xyl

Fuc

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

식물

특이당

제거

단백질

α
1,6

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

β
1,4

β
1,4

α
2,6

α
2,3

인간화

단백질

α
1,3

β
1,4

β
1,4

β
1,2

α
1,6

α
1,3

β
1,2

β
1,2

β
1,3

β
1,3

α
1,4

α
1,4

식물


단백질

1
단계
:
식물

특이적

glycosyltransferase


glycosidase


제거

2
단계
:
인간

glycosyltransferase


도입

1.
ER

retention

of

the

glycoproteins

2.
Suppression

of

glycocosyltransferases

and

glycocosidases

using

siRNA


3.
In

vitro

trimming

or

addition

of

the

sugar

residues

4.
Disruption

or

addition

of

glycocosyltransferases

and

glycocosidases

genetically


23

식물

생산

바이오

의약품의

안전성

제고를

위한

식물

N
-
glycan


인간화

GnTI

α1,3fucT

Man5

Man5Gn

MGnXF
3

MMXF
3

GnGn

GnGnXF
3

GnMXF
3

MGn

α1,3fucT

Hex

Hex

GnTII

Hex

Hex

αManII

β1,2xylT

β1,2xylT

1
단계
:
식물

특이적

glycosyltransferase


glycosidase


제거

GnTI

α1,6fucT

Man5

Man5Gn

MGnF
6

GnGn

GnGnXF
6

GnMXF
3

MGn

α1,6fucT

GnTII

αManII

GnTII

β1,4galT

2
단계
:
인간

glycosyltransferase


도입

α
1,6

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

β
1,4

β
1,4

α
2,6

α
2,3

인간화

단백질

α
1,3

β
1,4

β
1,4

β
1,2

α
1,6

α
1,3

β
1,2

β
1,2

β
1,3

β
1,3

α
1,4

α
1,4

식물


단백질

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

식물

특이당

제거

단백질

Martin
Gutternigg

et al., J. Biol. Chem.


(2007) 282
,
27825

27840,

Richard Strasser et al., Plant Physiol.

(2007) 145, 5
-
16

24

In
planta

protein
sialylation

through
overexpression

of
the respective mammalian pathway

J
Biol

Chem. (2010) 285:15923
-
30.

A. Castilho and R. Strasser
et al
.,

UDP
-
GlcNAc

ManNAc

ManNAc
-
6
-
P

NeuAc
-
9
-
P

Neu5Ac

GNE

GNE

NANS

NANP

CMAS

CMP
-
Neu5Ac

CST

Glycoconjugate

Sialoglycoconjugate

ST

GalT

Golgi

Nucleus

CTP

PEP

ATP

Cytosol

CMP
-
Neu5Ac

Isolation of T
-
DNA inserted Arabidopsis Mutants for
Humanized N
-
glycosylation

WT

fuct2

WT

fuct1

WT

xylt

WT

galt

WT

fuct3

WT

hex1

WT Ho

F + R

R + LB

WT

hex2

WT Ho

F + R

F + LB

WT

hex3

WT Ho

F + R

R + LB

25

Generation of Double Mutants by Crossing the
Single Mutants

fuct1
x
xylt

fuct1
x
fuct2

fuct1
x
fuct3

fuct2
x
fuct3

fuct2
x
galt

xylt
x
fuct3

fuct1
x ga
lt

fuct2
x xy
lt

fuct3
x ga
lt

galt
x xy
lt

26

Generation of Triple and Quadruple Mutants by
Crossing the Single Mutants

WT

fuct1

/
fuct2
/
xylt

WT

fuct1

/
fuct2
/
fuct3
/
xylt

27

Generation of the Quintuple by Crossing the
Mutants

WT

fuct1/fuct2/fuct3/
xylt
/
galt

28


1,3
-
fucosyltransferase I, II

β
1,2
-
xylosyltransferase

N
-
acetylglucosaminyltransferase I I

(GnT
-
II)

N
-
acetyl

glucosaminidase

I, III

(
Paucimannose

type N
-
glycan
)

UDP
-
Gal

Transporter


1,2
-
manno獩da獥s䤠⡍an䤩

N
-
acetylgluco獡minyltran獦era獥 䤠

(䝮T䤩

啄U
-
䝬cNAc

Tran獰orter


-
m
anno獩da獥s䥉
Ian䥉I

β
1,3
-
g
altosyltransferase (GalT)


1,4
-
fuco獹ltran獦era獥s

Analysis of
N
-
glycans

in the Double, Triple and
Quadruple Mutants

Concanavalin

A

Anti
-

HRP

Anti
-

fucose

Anti
-
xylose

Coomassie


29

30

Progress of the Arabidopsis Mutant Crossing

Double mutants

Note

Double mutants

Note

1

α
1,3
-
fuct1/
α
1,3
-
fuct2

O

6

α
1,3
-
fuct2/
α
1,4
-
fuct

O

2

α
1,3
-
fuct1/
β
1,2
-
xylt

O

7

α
1,3
-
fuct2/
β
1,3
-
galt

O

3

α
1,3
-
fuct1/
α
1,4
-
fuct

O

8

β
1,2
-
xylt/
α
1,4
-
fuct

O

4

α
1,3
-
fuct1/
β
1,3
-
galt

O

9

β
1,2
-
xylt/
β
1,3
-
galt

O

5

α
1,3
-
fuct2/
β
1,2
-
xylt

O

Triple mutants

Note

1

α
1,3
-
fuct1/
α
1,3
-
fuct2/
β
1,2
-
xylt

O

2

α
1,3
-
fuct1/
β
1,2
-
xylt/
β
1,3
-
galt

O

Quadruple

mutants

Note

1

α
1,3
-
fuct1/
α
1,3
-
fuct2/
β
1,2
-
xylt/
α
1,4
-
fuct

O

2

α
1,3
-
fuct1/
α
1,3
-
fuct2/
β
1,2
-
xylt/
β
1,3
-
galt



Quintuple

mutants

Note

1

α
1,3
-
fuct1/
α
1,3
-
fuct2/
β
1,2
-
xylt/
α
1,4
-
fuct/
β
1,3
-
galt

O

Septuple

mutants

Note

1

α
1,3
-
fuct1/
α
1,3
-
fuct2/
β
1,2
-
xylt/
α
1,4
-
fuct/
β
1,3
-
galt/Hex1/Hex3



31

Construct for
human
β
1,4
-
galactosyltransferase and
α
1,6
-
fucosyltransferase expression in plant

pGreenII 0229::CaMV35S:
α
1,6
-
fucosyltransferase:nos/CaMV35S:
β
1,4
-
galactosyltransferase:nos


kanamycin

®

Bgl

II

Kpn

I

Sac

I

Stu

I

Bgl

II

Bgl

II

RB

nos
-
bar ®

nosT

LB

CaMV35S

CaMV35S

nosT

β
1,4
-
galT

α
1,6
-
fucT

Not

I

Xba

I

Spe

I

Hin
d III

Sph

I

Pst

I

Sal

I

Xba

I

Bam
H I

Sma

I

Pst

I

Eco
RI

Nco

I

Sal

I

BspLU11I / Nco

I

Molecular

farming
기술의

실용화



상용화를

위한

주요

핵심기반기술


32

α
1,6

β
1,4

β
1,4

α
1,6

α
1,3

β
1,2

β
1,2

β
1,4

β
1,4

α
2,6

α
2,3

인체

단백질

α
1,3

β
1,4

β
1,4

β
1,2

α
1,6

α
1,3

β
1,2

β
1,2

β
1,3

β
1,3

α
1,4

α
1,4

식물


단백질



N
-
glycosylation


인간화











단백질

고발현



분리

정제

기술

확립








Promoter Affinity tag




단백질

폴딩

증진

기술

개발




















Molecular

farming


핵심기반기술

단백질

quality control


unfolded protein response
연구의

필요성

33


Fig.
Immunodetection

of heavy and light chain of
wt
2G12
SEC
,
wt
2G12
KDEL
,
TKO
2G12
SEC
,
wt
HA78
SEC

and
TKO
HA78
SEC.
(
a) Three
-
second film exposure;
(
b) 3
-
min film exposure.
wt
2G12
SEC

(lane 1);
wt
2G12
KDEL

(2);
TKO
2G12
SEC

(3);
wt
HA78
SEC

(4);
TKO
HA78
SEC
(5). HC, heavy chain; LC,
light
chain; arrow heads indicate
degradation products.


34

ER chaperone
들에

의한

분비단백질
,

막단백질

등의

폴딩



조절

BiP

BiP

Oligosaccharyl

transferase

Dolichol

oligosaccharide

Cytosol

ER lumen

SH

PDI

Calnexin

Calreticulin

Membrane
-
spanning

α

helix

Completed

monomer

Luminal

Α

helix

Trimer

Molecular Cell Biology, Sixth Edition

2008
W.H.Freeman

and Company

Protein disulfide

isomerase (PDI)


Calnexin, calreticulin:


Protein quality control

BiP(Binding Protein;


= Hsc 70)

35

Quality Control

of
Glycoprotein in the ER

P

P

Dol

Legend

α
1,2
-
Man

α
1,3
-
Man

α
1,6
-
Man


β
1,4
-
Man

β
1,4
-
GlcNac

α
1,2
-
Glc

α
1,3
-
Glc

Glc I

Glc II

Glc II

Glc II

Glc Trans (Parody enzyme)

(only acts on unfolded glycoproteins)

Calreticulin

KDEL

KDEL

Protein Folding

(other chaperonins)

Protein Folding

(other chaperonins)

Glc II

Glc II

Calnexin

ER membrane

Endoplasmic

Reticulum

Cytoplasm

Anteriograde

Transport

to Golgi

ER
-
associated
degradation (ERAD)

eIF2


36

Unfolded Protein Response Signaling Pathway

P

P

P

P

ATF6

IRE1

PERK

ER stress sensors

P

S1P

Translation block

ATF4

p50ATF6

splicing

TRAF2

Ask1

JNK

CRE

ATF6 site

ERSE

ER stress

Accumulation of unfoled proteins

:unfolded proteins

:GRP78/Bip

Nucleus

ER
-
chaperone

ER

XBP1

XBP1

XBP1

ATF6

ATF6

ATF6

ATF6

Induction of

molecular chaperones

ATF4

Ire1:
Cell, 132, 24
-
26 (2008)


PERK :
Proc Natl Acad Sci
USA
,
99
,

15920

15925
(
2002
)


ATF6:
Cell, 107, 881

891
(2001)


XBP1:
Nature, 463, 1092
-
1095
(2010)


eIF2
α
:
Science, 307, 935
-
939,
(2005)


ATF4:
Cell Research
,
20, 174

184 (2010)


JNK :
Science, 287, 664
-
666
(2000)

Unfolded Protein Response Signaling Pathway in
Plant

37

ER stress, Unfolded proteins

S. cerevisiae

Ire1p

Transcriptional upregulation of ER chaperones, glycosylation/modification,

and secretion pathway genes

Plants

Mammals

ATF6

Splicing of ATF6
cytosolic domain

PERK

elF
-
2
α

phosphorylation

Attenuation of
protein synthesis

Ire1
α

Ire1
β

Other Ire1


family members?

Up
-
regulation of
Xbp
-
1

mRNA

ATF6???

Ire1
α

Ire1
β

HAC1

mRNA
splicing

Xbp
-
1

mRNA???

Xbp
-
1

mRNA
splicing

-

Tm


+ Tm

Hac1

:
Nature, 457: 736
-
740 (2009), Nature, 457: 668
-
669 (2009),
Nature, 457: 687
-
693
(2009)

38

Molecular farming is a
synthetic biology

using
plants

Synthetic Biology


Design and construction of new biological parts, devices, and systems


Redesign of existing, natural biological systems for useful purposes

Promoter

Signal sequence

Translational enhancer

Fusion tag

Target gene

T
-
DNA

Host development

Plant
-
specific sugar deficient host

Humanized
glycan

producing host

Quality control

Unfolded protein response

Synthetic Biology

Transformation

Cultivation

Purification

39

Gaucher

Disease
,
the most prevalent
Lysosomal

Storage
Disorder (LSD)



Type 1

Type 2

Type 3

Frequency

General population:

1 in 50,000

Ashkenazi Jewish:

1 in 450

<1 in 100,000

<1 in 100,000

Neurological effect

Nonneuronopathic

Acute Neuronopathic

Chronic Neuronopathic

Symptom onset

Any age

First year of life

Childhood

Disease Course

Progressive

Rapidly progressive

Progressive

Phenotype

40

Clinical Manifestations of
Gaucher

Disease and
Glucocerebroside

Accumulation

Found by
Phillipe Gaucher

in 1882

Gaucher

patient’s

lysosomes

Full complement of

digestive enzymes

except glucocerebrosidase

glucocerebroside

Digestive

vacuole

Incomplete

digestion

Residual vacuoles
accumulate
because
glucocerebroside
can’t be digested


No exocytosis


41

Enzyme Replacement Therapy of
Gaucher

Disease:

Protein

Uptake

of

Macrophages

via

Mannose
-
Specific

Endocytosis

Receptor

Neuraminidase

GC

β
-
Galactosidase

β
-
N
-
acetylglucosaminidase

GC

Glucocerebrosidase

N
-
acetylglucosamine

Mannose

Galactose

N
-
acetylneuraminic acid

Fucose

GC

GC

GC

(60,43 kDa)

(67,00 kDa)

Developed by

Roscoe O. Brady

in 1973

1.
Ceredase
®
:

a

placentally
-
derived

form,

1991

2.
Cerezyme
®
:

a

recombinant

form,

1994

3.
Approved

for

type

1

Gaucher

disease

4.
Targeting

to

the

appropriate

cells

is

difficult


5.
Does

not

provide

a

long

life

cure

6.
Too

expensive
.

CR

FNII

TM

CT

4

5

7

1

3

2

6

8

42

Cerezyme

(a
Glucocerebrosidase

Produced by
Genzyme

corp.) Market


Genzyme

is

currently

the

only

provider

of

a

treatment

for

Gaucher’s

disease
.



Genzyme’s

behavior

in

the

Cerezyme

market

appears

to

be

very

close

to

a

perfect

monopoly
,

namely,

the

company

can

charge

its

profit
-
maximizing

price
.



The

market

for

Gaucher’s

disease

treatment

has

always

been

a

lucrative

market
,

and

other

companies

have

tried

to

develop

effective

treatments
.


The

Cerezyme

patent

expired

in

2001

but

its

manufacturing

method

is

patented

until

2011

and

its

composition

until

2013

(
Genzyme

corp
.
,

2003
)
.



The

average

wholesale

price

for

200

unit

vial

is

$
790

and

the

average

cost

per

patient

is

$
175
,
000

(
1


7


5


만원
)

annually
.

Genzyme

corp.

Cerezyme

Feb. 26, 2010

D
evelopment of Mannose
-
type N
-
glycan

Production Plant

43

gnt1

Golgi

Mammalian Golgi

(High mannose type N
-
glycan
)


1,2
-
mannosidase I (ManI)

N
-
acetylglucosaminyltransferase I

(GnTI) defect


UDP
-
GlcNAc

Transporter


1,2
-
manno獩da獥s䤠⡍an䤩

N
-
acetylgluco獡minyltran獦era獥 䤠

(䝮T䤩

啄U
-
䝬cNAc

Tran獰orter


-
m
anno獩da獥s䥉
Ian䥉I


1,6
-
fuco獹ltran獦era獥

N
-
acetylgluco獡minyltran獦era獥 䤠I

(䝮T
-
䥉I

β
1,4
-
g
altosyltransferase (GalT)

Sialyltransferases (SAT)

(Complex type N
-
glycan)

CMP
-
NeuAc

synthetase

UDP
-
Gal

Transporter

: Sialic acid

: Galactose

: Xylose

: Folded peptide chain

: Fucose

: GlcNAc

: Mannose

: Phosphoric acid

Plant Golgi


1,3
-
fucosyltransferase I, II

β
1,2
-
xylosyltransferase

N
-
acetylglucosaminyltransferase I I

(GnT
-
II)

N
-
acetyl

glucosaminidase I, III

(Paucimannose type N
-
glycan)

UDP
-
Gal

Transporter


1,2
-
mannosidase I (ManI)

N
-
acetylglucosaminyltransferase I

(GnTI)

UDP
-
GlcNAc

Transporter


-
m
anno獩da獥s䥉
Ian䥉I

β
1,3
-
g
altosyltransferase (GalT)


1,4
-
fuco獹ltran獦era獥s


1,3
-
fuco獹ltran獦era獥s䤬I䥉

β
1,2
-
xylosyltransferase

N
-
acetylglucosaminyltransferase I I

(GnT
-
II)

N
-
acetyl

glucosaminidase I, III

UDP
-
Gal

Transporter


-
m
anno獩da獥s䥉
Ian䥉I

β
1,3
-
g
altosyltransferase (GalT)


1,4
-
fuco獹ltran獦era獥s

Isolation of
Arabidopsis thaliana N
-
acetyl
-
glucosaminyltransferase

I (
gnt1
) Mutant

α
(1,3)

α
(1,6)

β
(1,4)

β
(1,4)

α
(1,3)

α
(1,6)

Protein

44

Expression of
Glucocerebrosidase

in
Arabidopsis

45

pGreenII

0179::
Cruciferin

promoter
-
signal sequence:Glucocerebrosidase
-
6His

LB

Cruciferin

p
romoter:
signal

sequence

RB

Cruciferin

Terminator

Glucocerebrosidase
-
6His

CaMV

35S
hygromycin

®


kanamycin

®

Expression of
Glucocerebrosidase

in

Arabidopsis seed

46

Coomassie

Anti
-
GC

Coomassie

Anti
-
GC

Coomassie

Anti
-
GC

Expression of
Glucocerebrosidase

in

Arabidopsis seed (3 mg/g TSP)

47

200 U vial = 6.5 mg =
$790

Glucocerebrosidase

(GC) expression in
Arabidopsis

seeds

Glucocerebrosidase

in TSP (w/w) = 3 mg/g = 0.3%

Protein contents of Arabidopsis seeds (w/w) = 23%

Glucocerebrosidase

in seeds (w/w) = 0.7 mg/g = 0.07%

Thank you

for your attention!

48

Gyeongsang

National University

Plant Biotechnology Lab.
(
http://pbtl.gnu.ac.kr
)


Wahyu
Indra
Fanata


In Jung
Jung


Bo
Hwa

Son


Jae Yong
Yoo


Je
Hein Kim


Rikno Harmoko


Ki

Seong

Ko


Sang
Yeol

Lee,
Ph.D.



Kyun

Oh Lee, Ph.D.
(
leeko@gnu.ac.kr
)