ELP-FUSION TECHNOLOGY FOR BIOPHARMACEUTICALS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 211

was useful for the production of safe and effective vaccin es,
fulfilling the approval requireme nts of the regulatory system
[94]. Re cently, Pfiz er has acqui red the exclusive wor ldwide
licensing rights to com mercial ize Protalix BioTher apeutics
Inc.'s taligluc erase alfa, a plant cell expressed form of
glucocer ebrosida se for the treatme nt of Gaucher 's dise ase
(www.protal ix.com). This drug is produc ed in a cont ained
disposable bioreac tor system with suspensi on-cu ltured car-
rot cells and is optimisti cally predi cted to receive FDA
approval, mak ing it the first plant- made pharmac eutica l
[95]. A recent press release announc ed that Bayer started
a clinical Phase I study wi th pers onalized vaccines from
tobacco plan ts, produced with the magnICO N 1 system [96],
for treatme nt of non-Hodgk in' s lymphom a. 1
The com bination of plan t-based expression and the ELP-
fusion techno logy repr esents an auspi cious chanc e to solve
aforement ioned challenges of traditi onal protein produc tion
systems. Alread y 14 years ago, first attempts were under-
taken to use plan t-based expressio n systems for the produc -
tion of the b ioelastic polymer wi th 121 repeats of the amino
acid seque nce Gly-Val-Gly- Val-Pro either in cultured cells
of tobacco [97] or in stable nucl ear transge nic tobacco plan ts
[98]. Despite a marginal accumulat ion of polymer protein
(0.01- 0.05% of total soluble protei n, TSP), (Gly-Val-Gly -
Val-Pro) 121 was pu rified usin g the temperat ure-depe ndent
aggregation, but only a portion of polymer prot eins
(

from bacterial production syst ems to eukar yotic expression
system s. Furth ermore, biocomp atibility of spid er silk ela stin
was demon strated by growth of anchorage -depe ndent mam-
malian cells (CHO K1, human chondro cytes) on SO1-
100xEL P coated cultur e plates compare d to conventional
coating s [100]. ELPylated SO1 was used for the prepa ration
of laye rs and foils, whi ch were more elastic and harde r than
therm oplastic polym ers currently used for industria l appli-
cations [102,1 03]. A second spider silk protei n, the native
major amp ullate spidroin protei n 2 (MaS p2) from Nephila
clavipe s was also fused to an ELP part consist ing of 27
repeats of Val-P ro-Gly-Xaa -Gly and expresse d in toba cco.
Despite a reported 60-fo ld higher level of reco mbinant
protei n in transge nic plan ts, the act ual accumul ation was
about 0.0125% TSP for MaSp2 and 0.75% TSP for MaSp2-
ELP, respect ively [104].
The un expecte d enhanc ement in reco mbinant protein
yield due to the C-te rminal ELP part was further confirm ed
for seed-pro duced single-cha in Fv (scFv ) ELP fusions. Here,
the ELP-fus ion strategy resulted in a 40-fold increas e in
scFv accumul ation with levels appro aching 25 % of total
soluble seed protein [105] . Plant seeds are partic ularly
attract ive for the produc tion of recombinan t antibodi es
becau se they remain stab le and functional even if matur e
seeds are stored at ambien t temperat ure for several year s
[106,1 07]. The scFv- ELP fusi ons from tobacco seeds exhibit
similar binding behavior to the correspo nding unfused anti-
bodies indicati ng that ELP do es not abrogate antigen bind-
ing [105] . A similar appro ach was desi gned by Joens uu and
collea gues to produc e scFvs agai nst the foot-and-m outh
disease virus (FMDV) in N. tabacu m as ELP-fus ion protei ns
with 28 repeats of the pent apeptid e Val-Pro-Gly- Val-Gly
[108]. Furtherm ore, a toba cco etch virus (TEV ) cleavage
side was include d between the scFv and the ELP part for the
removal of the ELP tag. The highe st scFv accumul ation level
was 0.08% TSP (transien t expressi on) and 0.8% TSP for
stable expression, resp ectively. The ITC process in combi-
nation with StrepII affini ty chromatogr aphy was used to
purify 1.5mg recombinant scFv-ELP fusions from 660mg
leaf material. After overnight cleavage the ELP part and the
protease were removed by sepharose capture using the His-
tag on the TEV protease and the StrepII-tag on the ELP. The
binding of purified scFv proteins prior to and after ELP
removal to FMDV was investigated and a higher binding of
scFv without ELP was observed [108].
The reputation of ELP tags have been increased due to
their salt- and temperature-dependent phase transition,
which can be used for nonchromatographic separation of
recombinant proteins, a significantly increase in the accu-
mulation of the fusion protein and their biocompatibility.
Therefore, the ELP-fusion strategy is a promising approach
for the in planta production of various pharmaceutical
proteins. Among the plant-based production of ELPylated
antibodies or antibody derivatives, vaccines and other proteins

0.003-0.0 3% o f TSP) was extractabl e resulting in a yield
between 0.5 and 5 m g of polymer p rotein from 1 g of fresh
weight of leaf tissue [98] . Th e unexpected low level o f
production was explained on the one side with the proka ry-
otic-pref erred polymer and on the othe r side with the
availabil ity of the amino acids glyc ine, valine, or proline
[98]. To overco me this problem , (Gly-Val-Gly-Val-Pro) 121
was expressed in chloroplast s du e to the pres ence of certain
amino aci d pools . Des pite of the presence of higher polymer
transcrip t levels in transplastom ic plants, the protei n expres-
sion was quite low, which was justifi ed with glycine as the
limiting factor for productio n in chlo roplasts [99].
In 2004, the traditi onal ELP-fus ion strategy from Meyer
and Chilkoti was transferr ed to the plant biotech nology field
to produc e spid er silk elastin in the endoplasm ic reticulum
(ER) of tra nsgenic tobacco and potato plants, and further-
more to purify the reco mbinant protei n by a simpl e method
using heat treat ment and ITC (Fig ure 14.4A ) [100] . Gen er-
ated transge nic plants accumul ated recombinan t synthet ic
spider silk protein SO1 [101] fuse d with the ela stic bio-
polymer 100xELP from 0.5% up to 4% of TSP. Unexpect-
edly, fusi on of SO1 with 100xEL P resu lted in a twofold
increase in the accumulat ion level com pared to the naked
SO1 protei n (0.5-2% [101]. This was the first report on the
successf ul purificatio n of reco mbinant ELP-fus ion proteins
from crude leaf extracts and confirm ed the port ability o f ITC

1 http://www.icongenetics.com/html/5954.htm