cardineau guy pep talk 11, jan 12,2012
TRANSCRIPT
Guy A. Cardineau, Ph.D.
Higher Accumulation of
F1-V Recombinant Fusion Protein in Plants After
Induction of Protein Body Formation
Director, Centro de AgrobiotecnologíaDepartamento Agrobiotecnología y AgronegociosTecnológico de Monterrey, Campus Monterrey
ASASU Centennial Professor, Emeritus
Research Professor, Emeritus & Faculty Fellow
Center for Infectious Disease and Vaccinology
The Biodesign Institute,
The School of Life Sciences and
The Sandra Day O’Connor College of Law
Arizona State University
Biotechnology Drug Approvals 1982-2008While the number of approved biotech-based products approved per year is variable, the trend is upward. Biotechnology drugs appear the fastest-growing sector for drug development, and it is predicted that biotech drugs will comprise over 50% of all drug approvals by 2015 and more than 75% by 2025. These predictions are supported by the expected benefits of increased understanding of drug targets and the molecular and genetic bases of disease, as well as the declining conventional small-molecule drug pipelines in most major pharma companies. BioWorld Today Sept 1,2009
The table to the left represents information from an article published in BioWorld Today in late August 2009, written by Michael Harris,
2
late August 2009, written by Michael Harris, Executive Editor, about the top 25 biotech drugs currently on the market. The data provided includes revenues for each of these biotech drugs in 2008 (>$70B US), the date each drug product was first approved by the FDA and when patents protecting each drug are due to expire. It should be kept in mind that one feature of all these drugs is that they have been approved for more than one ndication; Harris reports that Genentech's Avastin is being tested in more than 450 clinical trials for treating more than 30 different types of cancer. It should also be kept in mind that 7 of the 25 "biotech" drugs are small molecules, and another 6 are antibodies.
Historically, Plants Have Been Routinely Used to Produce Pharmaceuticals, Naturally
� Global over-the-counter sales of plant-derived drugs are estimated at $40 billion per yearWell established regulatory systems are in place for these products
� Estimated one-quarter of the prescription drugs sold in the US, Canada and Europe contain active ingredients derived from plants
� Tens of thousands of plants are used for medicinal purposes
� Well established regulatory systems are in place for these products
Drug/Chemical Action/Clinical Use Plant Source Cocaine Local anaesthetic Erythroxylum coca Codeine Analgesic Papaver somniferum Digitalin, Digitoxin Cardiotonic Digitalis purpurea Quinine Antimalarial Cinchona ledgeriana Taxol Antitumor agent Taxus brevifolia Vinblastine, Vincristine Antitumor, Antileukemic Catharanthus roseus
SUMMARY from Large Scale Biology, Inc.
• Hormones and immune modulators
• Monoclonal antibodies - IgG
• Subunit vaccines
• Enzymes
Classes of New Protein Drug ProductsClasses of New Protein Drug Products
Production Systems in UseProduction Systems in UseProduction Systems in UseProduction Systems in Use• Bacterial fermentation
• Mammalian cells in fermentation
• Yeast
• Insect cells (GSK’s cervical cancer vaccine; 2005/6)
• Green plants – Stable and Transient Transformation,
Whole Plants and Plant Cells
One approved product in the market in plant cells
• Bacterial fermentation
• Mammalian cells in fermentation
• Yeast
• Insect cells (GSK’s cervical cancer vaccine; 2005/6)
• Green plants – Stable and Transient Transformation,
Whole Plants and Plant Cells
One approved product in the market in plant cells
Early Patent Filings on
Plant Made
Pharmaceuticals
5
Original Concepts of Therapeutic Protein,Vaccine Antigen, and Antibody Expression in Plants
Dow AgroSciences/ASU collaboration
developed a Newcastles Disease Virus
subunit vaccine in tobacco NT1 cells.
United States Patent 7,132,291, Cardineau, et al., November 7, 2006 (Canadian counterpart CA 2524293)
Vectors and cells for preparing immunoprotective compositions derived from transgenic plants AbstractThe invention is drawn towards vectors and methods useful for preparing genetically transformed plant cells that express
immunogens from pathogenic organisms which are used to produce immunoprotective particles useful in vaccine preparations. The
invention includes plant optimized genes that encode the HN protein of Newcastle Disease Virus. The invention also relates to
methods of producing an antigen in a transgenic plant.
WHY ORALLY DELIVERED PLANT-MADE VACCINES?
� Plant-derived vaccines are cost-effective andstable at room temperature.
� Plants provide both an encapsulated antigenand an oral delivery system that stimulatesthe mucosal immune system resulting in bothsecretory and circulating antibodies.
� The mucosal immune system is the first lineof defense against most pathogens.
� Oral vaccines are potentially safer, require noneedles and may not require trained medicalpersonnel to administer.
� Several Phase I Human Clinical Trials with plant-made vaccines have been run resulting in positive immune responses.
WHY INCREASE F1-V FUSION PROTEIN ACCUMULATION IN PLANTS?
�Our primary objective is to produce plant-derived heat stable vaccines that can be delivered orally.
�We have been using F1-V, a fusion between two antigens from the plague bacterium Yersinia pestis, as our model antigen in production improvement studies.pestis, as our model antigen in production improvement studies.
�We are assessing parameters that affect expression of F1-V fusion protein in plants and plant cells to be used as both a production and delivery system of vaccines and potentially other biopharma proteins.
�High antigen accumulation is required to compensate for partial proteolysis in the gut upon oral delivery.
Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• To date, most efforts have focused on increasing protein
synthesis.
– enhanced transgene expression can be obtained by optimizing
regulatory elements including stronger promoters, transcriptional enhancers, translational enhancers, alternative polyadenylation signals,
using synthetic genes with codons that have been optimized for gene
expression in target plants, overcoming RNAi and silencing
• Unfortunately, high transgene expression does not always
guarantee high levels of recombinant protein accumulation
since proteins may be expressed successfully but
subsequently degraded.
• It has been demonstrated that post-synthesis and/or post-
secretion instability and degradation are critical factors
contributing to low foreign protein yield.
25000
30000
35000
preboos t
pos tboos t
ANIMAL TRIALS: PRIME-BOOST STRATEGY
PRIME: s.c. 15 µg bacterially derived F1-V
BOOST: 2 g non-transgenic tomato (n = 5) on days
BOOST: 2 g F1-V transgenic tomato (n = 6) on days 21, 28, 35 (300 ug) and 42 (1200 ug)
[Ug/m
l]
250
300
350
preboost
postboost
[Ug/m
l]
0
5000
10000
15000
20000
25000
30000
35000
F1-spec if ic IgG1 V -spec if ic IgG1
preboost
postboost[Ug/m
l]
F1-specific IgG1 V-specific IgG1
0
50
100
150
200
250
300
350
F1-specific IgG2 V-specific IgG2
preboost
postboost[Ug/m
l]
F1-specific IgG1 V-specific IgG1
0
5000
10000
15000
20000
F1-spec if ic IgG1 V -spec if ic IgG1
Combined F1-V and V-specific IgG1 titerscorrelate with protection in mouse model(Williamson et. al., Clin. Exp.Immunol., 1999, 116; 107-114.)
tomato (n = 5) on days 21, 28, 35 and 42)
F1-specific IgG1 V-specific IgG10
50
100
150
200
F1-specif ic IgG2 V-specif ic IgG2F1-specific IgG2a V-specific IgG2a
CHALLENGE (s.c. 20 LD50 Y. pestis)
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Days post-infection
% s
urv
ival
TG
WT
CONTROLSChallenge of the vaccinated mice
with s.c. Y. pestis
Alvarez & Cardineau Biotechnology Advances
2010, 28 (1): 184-196
% o
f su
rviv
al
Days post-infection
CHALLENGE (s.c. 20 LD50 Y. pestis)
12
Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• There are several possible sites and mechanisms of foreign
protein degradation in plants. Cytoplasmic proteases contribute
significantly to product losses within plant cells.
• Proteolytic degradation of foreign proteins can be minimized by
targeting synthesis to the endoplasmic reticulum (ER) rather than
the cytosol, but this doesn’t always work. the cytosol, but this doesn’t always work.
– ER retention of soluble transport-competent proteins is inducible by the
carboxy-terminal retention/retrieval signal KDEL or HDEL, which is
recognized by a receptor located in the Golgi complex.
– Upon binding, the receptor retrieves C-terminal tagged proteins back into the
ER. Localization within the ER via the addition of KDEL or HDEL increases
the accumulation of foreign proteins in transgenic plants.
– However, the ER retention via KDEL is mediated by a KDEL receptor.
When the receptor is saturated with KDEL ligands, the KDEL-tagged recombinant protein either secretes or is transported to the lytic vacuole
Protein accumulation in plant tissues reflects a
balance between protein synthesis and degradation
• Some KDEL-tagged recombinant protein can be also misfolded
and delivered for degradation through an ER-dependent
mechanism named ‘‘unfolded protein response’’ or UPR, which
functions for both endogenous or heterologous proteins
• The K/HDEL system is common to all eukaryotes, but plants can
use a different ER localization system in seeds consisting of
specialized organelles called protein bodies (PB), which stably specialized organelles called protein bodies (PB), which stably
accumulate seed storage proteins within the ER.
• The maize 27 kD γ-zein seed protein is not secreted even though
it bears an N-terminal signal sequence and lacks a canonical
KDEL/HDEL ER-retention signal; it is able to form ER-localized
PB not only in maize endosperm but also when expressed in
storage or vegetative tissues of transgenic Nicotiana tabacum,
Hordeum vulgare and Arabidopsis thaliana plants, respectively.
• PB formation can lead to higher protein accumulation in the ER
possibly because of the exclusion from the normal ER turnover
The Rules of Science
WHAT IS ZERA?
� Cereal grains have evolved to store large amounts of proteins:γ-Zein is the major storage protein in maize.
� Zera (γ-Zein ER-accumulating domain) is the N-terminalproline-rich domain of γ-zein that is sufficient to induce theassembly of protein bodies.
� Zera adopts an extended helix conformation where polar� Zera adopts an extended helix conformation where polarresidues (histidines) are located on one side of the helix andhydrophobic residues (leucines and valines) on the oppositeside of the helix.
� This conformation provides high solubility in aqueous mediaand the ability to self-assemble both in hydrophobic andhydrophilic environments.
� The Zera domain retains its ability to developprotein bodies after being fused with an exogenousprotein of interest.
� Zera contains two targeting signals:
ZERA PROTEIN BODIES
Organelles surrounded by a membrane derived from the ER. Organelles surrounded by a membrane derived from the ER.
� Zera contains two targeting signals:
1- A signal peptide that internalizes Zera fusionprotein inside the ER
2- The Zera domain itself that oligomerizes coatingthe ER membrane and inducing the protein bodyformation.
The basis of Zera® technology
Nature knows how to assemble and store proteins in seeds in Protein Bodies
Zera® is a natural peptide from a corn storage protein, γ -Zein, that has assembling properties
Zera® can be used as a tag, in fusion with the protein of interest
1. Protein bodies are obtained
directly from the biomass
Zera ®Recombinant
product
© ERA Biotech SA | January 12 18
� Effects on expression level
� Formulation / Protection / Stability
� Activity, even in fusion, even assembled
directly from the biomass
3. When needed, a cleavage
can be done by proteases or
inteins*
4. Pure protein is obtained by
classical chromatography
technique
2. Solubilisation under mild
conditions
The benefits of Zera induced protein bodies (PBs)
� Zera fusion proteins inside PBs escape the ER degradation
pathway allowing higher accumulation rates.
� The accumulation of the Zera fusion proteins in PBs also
protect the plant cell from toxic proteins.protect the plant cell from toxic proteins.
� Post-translational modifications of Zera fusion proteins inside
PBs: ER classical processing (N-glycosylation). Absence of
Golgi complex glyco-modifications.
� The easy isolation of the protein body-like organelles makes
them an extraordinary enrichment tool.
20
TRANSIENT EXPRESSION OF F1-V FUSION PROTEIN IN N. benthamiana
pCaSFV
5’ CsVMV3’ Ag7 5’ NOSLB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB
pCaSFV
5’ CsVMV3’ Ag7 5’ NOSLB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB5’ CsVMV3’ Ag7 5’ NOSLB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB5’ CsVMV3’ Ag7 5’ NOSLB RB
NPT2 F1-V fusion
5’ vspA SP
3’ vspB
pCFV
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
pCFV
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT210 2
35S
:Zera
-F
1-V
35S
:F1-V
CsV
MV
-F1-V
CsV
MV
-SP
-F
1-V
ng bacterialrF1-V
W.TRB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7
RB5’ CsVMV 3’ vspB5’ NOS
LBF1-V fusion3’ Ag7 NPT2
p35SF1V
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusion
TEV-5’ UTR
p35SF1V
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusion
TEV-5’ UTR
RBRB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusion
TEV-5’ UTR
5’ CaMV35S 3’ vspB3’ Ag7 5’ NOSLB
Bar F1-V fusion
TEV-5’ UTRp35S:Zera-F1V
TEV-5’ UTR
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusionZera
p35S:Zera-F1V
TEV-5’ UTR
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusionZera
TEV-5’ UTRTEV-5’ UTR
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusionZera
RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS
LBBar F1-V fusionZera
10 2 2
V
W.T.
Zera-F1-V
(67 kDa)
F1-V(56 kDa)
Zera-F1-V
dimers
NT1 TRANSFORMATION: Zera-F1-V vs. F1-V
3-week old
Selection of the healthiest NT1 calli
3-week old calli
Liquid culture of NT1 cells
Freeze-dried NT1 cell culture.
Selection of the elite lines by Western-blot
8 weeks after transformation
200
250N
um
ber
of
calli re
co
vere
d
F1VLBA4404 / F1-V
NT1 TRANSFORMATION: ZERA-F1-V vs. F1-V
200
250N
um
ber
of
calli re
co
vere
d F1V
LBA4404/ ZeraF1V
LBA4404 / F1-V
200
250N
um
ber
of
calli re
co
vere
d F1V
LBA4404/ ZeraF1V
GV3101 /ZeraF1V
LBA4404 / F1-V
150
200
250N
um
ber
of
call
i re
co
vere
d F1V
LBA4404/ ZeraF1V
GV3101 /ZeraF1V
F1-V: 49 calli
Zera-F1-V:
2 calli
transformation
0
50
100
150
6 7 8 9 10 11 12 13 14 15
Time [weeks]
Nu
mb
er
of
calli re
co
vere
d
0
50
100
150
6 7 8 9 10 11 12 13 14 15
Time [weeks]
Nu
mb
er
of
calli re
co
vere
d
0
50
100
150
6 7 8 9 10 11 12 13 14 15
Time [weeks]
Nu
mb
er
of
calli re
co
vere
d
0
50
100
150
6 7 8 9 10 11 12 13 14 15
Time [weeks]
Nu
mb
er
of
call
i re
co
vere
d
GV3101 /ZeraF1V
EHAO105 / ZeraF1V
IMMMUNO-ELECTRON-MICROSCOPY OF ZERA-F1-V TRANSGENIC NT1 CALLI
Immunocytochemistry using anti-Zera or anti-F1-V antibody
F1-V FUSION PROTEIN
ACCUMULATION IN NT1 CALLI
0
5000
10000
15000
20000
25000
Zera-F1-V NT1 F1-V NT1
Ba
nd
in
ten
sit
y [
A.U
.]
F1-V fusion protein accumulation: >3X
higher in Zera -
F1-V than in F1-V NT1 calli
ALFALFA TRANSFORMATION: ZERA-F1-V vs. F1-V
ZeraF1-V
F1-V
1 month after transformation
ZERA-F1-V F1-V
day 0
1 month
19 elongated leaves (5% of explants)
144 elongated leaves (58% of explants)
ZeraF1-V
2 months
1 month
4-5 months
F1-V FUSION PROTEIN ACCUMULATION IN ALFALFA
F1-V fusion protein accumulation: >3X
higher in Zera-F1-V than in F1-V alfalfa.
0
5000
10000
15000
20000
25000
30000
Zera-F1-V F1-V
Ban
d in
ten
sit
y [
A.U
.]
ANALYSIS OF NT1 CALLI AND ALFALFA
BY F1-V SOUTHERN-BLOT ANALYSIS
ALFALFA
F1-V PROTEIN ACCUMULATION vs. GENE COPY NUMBER
Plant tissue
Line
Recombinant
protein
µµµµg F1-V/g TSP (*)
Gene copy #
(**)
Alfalfa leaves
A-Z51 Zera-F1-V 230 ± 20 3
A-Z35 Zera-F1-V 150 ± 10 n.d.
A-Z21 Zera-F1-V 160 ± 20 1
A-Z54 Zera-F1-V 200 ± 30 2
A-FV30 F1-V 55 ± 4 1
A-FV57 F1-V 58 ± 6 2
A-FV24 F1-V 50 ± 3 1
A-FV23 F1-V 55 ± 4 n.d.
NT1 calli N-Z1 Zera-F1-V 3800 ± 300 n.d.
N-Z5 Zera-F1-V 8500 ± 200 1
N-Z8 Zera-F1-V 6100 ± 500 3
N-Z4 Zera-F1-V 4900 ± 300 1
N-FV1 F1-V 1300 ± 100 1
N-FV4 F1-V 1700 ± 100 2
N-FV6 F1-V 200 ± 20 n.d.
N-FV28 F1-V 2000 ± 200 3
CONCLUSIONS
� The F1-V fusion protein accumulation in NT1 cells and alfalfa was at least 3X higher using Zera technology.
� The accumulation of F1-V in ER-derived PB-like structures induced by Zera was confirmed by EM.
� The regeneration of alfalfa or NT1 calli expressing Zera-F1-V was delayed compared to F1-V likely due to the PB-like formation.
� These results confirm the potential of Zera technology as a strategy to increase value-added proteins in plants.
Expression of ZERA®-GFP in N. benthamiana by agroinfiltration
P1 TnosTL enh
D35S
HcProHcPro
ZERA®-Gfp TnosTL enh
D35S
Zera®-GFP
© ERA Biotech SA | January 12 31
Zera®-GFP+
HcPro
Zera®-GFP
ZERA® technology can address unmet needs for therapeutic protein
developmentA highly efficient process is used to produce proteins based on ZERA® technology:
high expression levels and simple downstream process.Insect cells+X buffer
Homogenization by sonication
Centrifugation 10000g 20’
x3
H2O wash by sonication
Centrifugation 10000g 20’
x2
Sto
rPro
reco
ve
ry
© ERA Biotech SA | January 12 32
Potential for improved shelf-live under non-refrigerated conditions
Tobacco: protein extraction from fresh and dried leaves
20
Zera®EGF Zera®Ct Zera®T20
Tobacco: protein extraction from fresh and dried leaves
20
Zera®EGF Zera®Ct Zera®T20
1009994 92
87
68
26
100104
108 110
117
95
66
0
20
40
60
80
100
120
0 5 10 15 20 25
Re
ma
inin
g a
ct
(%)
Time (min)
Stability at 45ºC comm GOX
zGOX PBs
Glucoxidase (Gox) fused to Zera® and accumulated in StorPro® is
more stable at high temperature than wt Gox.
Immediatly extracted from fresh leaves
1wk 37ºC & 5 months RT storage
ZERA® technology can address unmet needs for vaccine development
ZERA® technology induce significant cellular and humoral immune responses.
The cellular immune responses elicited by vaccines based on ZERA® Technology confer protection and are cytotoxic.
Vaccines made with ZERA® technology have a positive immunomodulatory effect.Case studies: Zera®-E2 (Classical Swine Fever), Zera®-E7SH (Human Papilomavirus) and Zera®NP (Lymphocytic
Choriomeningitis virus)
14,00
16,001000000
Citotoxic immune response Challenge against LCMV infection
© ERA Biotech SA | January 12 33
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
%C
D8
+/IF
Nγγ γγ
+
Z-NP particles induce specific CD8 T-cells
in the absence of any extra-adjuvant
1
10
100
1000
10000
100000
PBS Zera-NP LCMV
Zera-NP StorPro bodies are efficient
immunogens against LCMV infection
*
Log
10
pfu
/gr
*
ZERA® technology can address unmet needs for vaccine development
Effective DNA vaccines could be also made using ZERA® Technology. Case studies: Zera®-E7SH (HPV) and Zera®NP (LCMV)
Log
10
pfu
/gr
1000
10000
100000
1000000
Challenge against LCMV infection
© ERA Biotech SA | January 12 34
Zera®-NP DNA vaccine protection is as efficient as LCMV in
challenge experiments
Log
10
pfu
/gr
1
10
100
1000
PBS Empty
vector
NP Zera-NP Zera LCMV
* *
S I2 I3 I4 P
PO
I
I2
10 %
20 %
I1
S
15 DAP
2 3 48 DAP 15 DAP
I1
I2
S10
20
Induced StorPro® in tobacco leafsNatural PBs in maize endosperm
Natural maize PBs and StorPro® bodies are dense organelles
© ERA Biotech SA | January 12 35
Ze
ra®
-PO
I
BiP
I2
I3
P
I4
27 %
42 %
56 %
BiP
27γ27γ27γ27γZ
PB
I3
I2
I4
ER
30
46
52
w/w
P
StorPro® bodies are highly packed assemblies which can be recovered
effiently by density gradients
Su
cro
se s
tep
den
sit
y g
rad
ien
t
H S I2 I3 I4 P
Density gradient
purification
H S
IF2
StorPro® bodies are dense organelles
© ERA Biotech SA | January 12 36
Su
cro
se s
tep
den
sit
y g
rad
ien
t
ZERA®-GFP
Centrifugation
80.000g 2h 4ºC
IF2
IF3
IF4
P
PBs
H H’ Pb S C RF
Zera-EGFZera-hGH
StorPro® bodies recovered by low-speed centrifugation
Some examples of Zera® fusion proteins recovered by low speed
centrifugation (1000-2500xg)
H H’ Sp W PB
© ERA Biotech SA | January 12 37
hGH
Preclarified homogenate (H); Clarified Homogenate (H’); Soluble protein discarded (Sp); Wash step (W);
StorPro fraction (Pb); Solubilized fusion protein (S); Cleavage step (C); Reverse phase purification (Rf)
There is no need of density gradient to recover StorPro® bodies in highly pure
fraction
StorPro® bodies recovered by low-speed centrifugation
Additional examples of Zera® fusion proteins recovered by low speed
centrifugation (1000-2500xg)
1. Zera
2. Zera-Bivalirubin
3. Zera-EGF
4. Zera-Insulin
5. Zera-hGH
6. Zera-Gfp
7. Zera-Gfp
8. Zera-Xylanase
1 2 3 4 5 6 7 8
© ERA Biotech SA | January 12 38
Value proposition: Zera® makes products better by accumulating more product
Industrial Enzymes
• Versatility to adapt to a broad spectrum of real industrial conditions.
• Readily immobilised purified enzymes while keeping the activity
• Capacity to produce multi-enzymatic StorPro bodies
0
50
100
150
Enz
Zera-Enz
Act
ivit
y
The Zera® technology improves the performance and properties of protein-based products and processes
– Versatility in terms of eukaryotic expression systems
– Versatility in terms of protein types (complex proteins, membrane proteins, etc)
© ERA Biotech SA | January 12 39
Vaccines for human and animal health
• Strong cellular response without adjuvants
• Efficient antigen presentation and protection
• Stable at room temperature
Therapeutic Products
• High activity performance of Zera® fusion peptides
• Incorporation of post translational modifications
• Multiple formulations and delivery formats from a single construct
Proliferation ZERA-Peptide
1 10 100 1000 100000
25
50
75
100
125
Cell line 1)
Cell line 2
nM
%P
roli
fera
tio
n
Acknowledgements
Boyce Thompson Boyce Thompson Boyce Thompson Boyce Thompson
InstituteInstituteInstituteInstitute
Dan Dan Dan Dan KlessigKlessigKlessigKlessig
Joyce Van EckJoyce Van EckJoyce Van EckJoyce Van Eck
TishTishTishTish KeenKeenKeenKeen
XiurenXiurenXiurenXiuren ZhangZhangZhangZhang
Wendy Wendy Wendy Wendy VonhofVonhofVonhofVonhof
Jason Jason Jason Jason EibnerEibnerEibnerEibner
NoreneNoreneNoreneNorene BuehnerBuehnerBuehnerBuehner
Bryan MaloneyBryan MaloneyBryan MaloneyBryan Maloney
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Chuck MihaliakChuck MihaliakChuck MihaliakChuck Mihaliak
Jennifer RiceJennifer RiceJennifer RiceJennifer Rice
Butch MercerButch MercerButch MercerButch Mercer
University of ArizonaChieri KubotaRyo Matsudo
4040
Hugh Mason Jilliane MillerCharles Arntzen Andrew Koons
Essential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and Collaborators
Arizona State UniversityArizona State UniversityArizona State UniversityArizona State University BiodesignBiodesignBiodesignBiodesign Institute at ASUInstitute at ASUInstitute at ASUInstitute at ASU
Cornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food Science Dow Dow Dow Dow AgroSciencesAgroSciencesAgroSciencesAgroSciences
Benchmark Benchmark Benchmark Benchmark BiolabsBiolabsBiolabsBiolabs US Department of DefenseUS Department of DefenseUS Department of DefenseUS Department of Defense
University of ArizonaUniversity of ArizonaUniversity of ArizonaUniversity of Arizona Science Foundation ArizonaScience Foundation ArizonaScience Foundation ArizonaScience Foundation Arizona
TecnologicoTecnologicoTecnologicoTecnologico de Monterreyde Monterreyde Monterreyde Monterrey FondosFondosFondosFondos ZHZHZHZH
FEMSAFEMSAFEMSAFEMSA
Butch MercerButch MercerButch MercerButch Mercer
Tec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de Monterrey
Israel RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael Ramirez Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Andrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea Martinez Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar
Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Federico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico Lopez Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia EchauriEchauriEchauriEchauriEchauriEchauriEchauriEchauri
Carlos Carlos Carlos Carlos Carlos Carlos Carlos Carlos OrigelOrigelOrigelOrigelOrigelOrigelOrigelOrigel Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez
Paulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina Calderon Cristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina Morales JoharisJoharisJoharisJoharisJoharisJoharisJoharisJoharis SalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgado Gonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo Mendoza
Miguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Miguel Miguel Miguel Miguel Miguel Miguel Miguel Miguel SuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuastegui
Cardineau Lab
Tec de Monterrey, Fall 2011
The Potential of Plants