Biomedical Engineering Reference
In-Depth Information
FIGURE 14.1 Protein purification of ELPs by inverse transition cycling (ITC) ELPs are soluble
(soluble ELPs, sELPs) at a temperature below their transition temperature (T t ) and become insoluble
when the temperature is increased above T t . The insoluble ELP-aggregates (iELPs) are precipitated by
centrifugation and thereby separated from contaminating proteins, such as cellular proteins. The
precipitate containing the iELPs is dissolved in buffer with a temperature below T t . Increased turbidity
of ELPylatedmini-sgp130 [113]was visualized using standard cuvettes below (A, sELPs) and above (B,
iELPs) T t . Source: Reprinted from Reference [9]. Copyright (2010), with permission from Elsevier.
ELPylation [9]. Initially, chemical methods were used for
the synthesis of ELPs [10]. Nowadays, applications of ELP-
fusion technology exclusively use genetically encoded ELPs
because the incorporation of a gene encoding the desired
ELP sequence into an expression vector allows the bio-
synthesis of a monodisperse polymer with the exact molec-
ular weight and the correct stereochemistry along with a
high yield [11]. Therefore, a synthetic oligonucleotide
(
chemical similarity of ELP to native elastin, which is
immunological inert and therefore attractive for biomedical
applications. Poly(Gly-Val-Gly-Val-Pro) has been used in a
number of tissues, tissue-fluid and blood tests as approved
by the relevant regulatory body of the United States [17].
Furthermore, in vitro and in vivo studies using poly(Val-Pro-
Ala-Val-Gly) did not reveal any evidence of cytotoxicity,
nonspecific depression of cellular respiration in macro-
phages, or general inflammatory responses [18]. Recently,
the effect of purified poly(Val-Pro-Gly-Xaa-Gly) on mouse
and bovine immune cells was investigated and no immu-
nomodulatory role was detected [19]. The design of ELPs
allows the manipulation of their stimulus-responsive prop-
erties and other physical or functional characteristics and has
led to biological applications of ELPs as structural tools for
recombinant protein purification and drug delivery, and as
material for tissue engineering. The following sections
highlight the use of ELPylation as a new tool to efficiently
purify recombinant proteins and summarize the application
of ELPs in medicine and biotechnology.
100 bp) designed to encode the desired ELP sequence
is oligomerized by recombinant DNA methods to create the
synthetic ELP gene. ELPs exhibit a thermally responsive
phase transition; they are monomeric and soluble at temper-
atures below the transition temperature (T t ), and aggregate
and become insoluble at temperatures above [8]. This
process, termed inverse phase transition, is reversible,
occurs within a 2-3 C range and can be monitored spec-
trophotometrically by measuring the turbidity of the protein
solution (Figure 14.1). This phenomenon can be explained
by the development of closer associations between single
b -spiral strands of ELP and the subsequent formation of
interstrand b -sheet structures that mediate close association
or aggregation of different ELP chains [12]. Temperature-
triggered insolubility and aggregation is reversed com-
pletely when lowering the temperature below T t , which
itself is defined by the combination of buffer composition,
salt concentration, ELP concentration, ELP chain length, the
degree of ionization of any functional side chains and the
guest residue Xaa, and the polarity arrangement along the
molecule [13-16]. These tuneable properties are transferred
to ELP-fusion proteins making ELPylation useful for bio-
logically inspired polymers that can respond to thermal and
other environmental signals. The exploitation of ELPs in
medicine and the suitability for in vivo applications is due to
the high level of biocompatibility of ELP. There exists a
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14.2 ELP-BASED PROTEIN PURIFICATION
Heterologous recombinant protein production in living pro-
karyotic and eukaryotic cells by biotechnological methods
provides the basis for therapeutic application of biologics in
autoimmune and chronic inflammatory diseases and cancer.
Biologics such as antagonistic monoclonal antibodies or
soluble cytokine receptors fused to the constant Fc part of an
IgG antibody were developed to inhibit molecules including
the inflammatory cytokines tumor necrosis factor or inter-
leukin-6 [20,21]. A cost-intensive issue of recombinant
protein expression methodology is the development of
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