Biomedical Engineering Reference
In-Depth Information
efficient downstream protein purification procedures. Tradi-
tional protein purification strategies such as size exclusion,
ion exchange, or affinity chromatography are rather cost
intensive. The commonly applied affinity chromatography
to purify recombinant proteins relies on a reversible highly
specific immobilization of the target protein. Antibodies
might be purified via their constant Fc parts using
Staphylococcus aureus Protein A affinity chromatography.
Nonimmunoglobulin target proteins might be fused to either
a carrier protein, such as Fc region of an immunoglobulin,
maltose-binding protein, glutathione S-transferase, or thio-
redoxin, to carrier peptides such as oligohistidine or the
FLAG-peptide (DYKDDDDK) allowing the purification of
the target protein almost independently of its physiochem-
ical properties. For therapeutic applications, these tags are
usually removed to obtain the therapeutic protein.
The thermally stimulated, reversible aggregation/preci-
pitation behavior of ELPs as carrier fused to a target protein
provides an alternative mode of protein purification and
avoids the rather cost-intensive approaches of other purifi-
cation strategies such as affinity chromatography. ELP-
based protein purification bypasses the need for affinity
chromatography; the complete process becomes simpler,
less expensive and is readily upgradable to a preparative
mode, which is discussed in the following section. Several
methods that allow the cleavage of the carrier ELP tag from
the target protein are also introduced.
The thermally stimulated purification of heterologous
expressed ELPs was first demonstrated for Escherichia
coli by McPherson and colleagues in 1996 [22]. Later on,
the thermally stimulated purification of recombinant carrier
ELP-fusion proteins by inverse transition cycling (ITC) was
achieved from bacterial cells [23] (Figure 14.2A). Here,
aggregation of ELPylated target proteins was achieved by
raising the temperature above T
t
. ELPylated target proteins
were pelleted by subsequent centrifugation. The resulting
supernatant, containing the undesirable host proteins, was
removed and the remaining recombinant ELP-target protein-
containing pellet was dissolved in low-salt buffer at a
temperature below T
t
. This strategy leads to an almost
complete purification of the ELP-target proteins. In this
example from Meyer and Chilkoti [23], the synthetic ELP
component of the fusion protein was an oligomerized series
of Val-Pro-Gly-Xaa-Gly pentapeptides, where Xaa pos-
sessed a 5:2:3 ratio of Val:Ala:Gly, engineered to produce
a specific T
t
. It became evident that the ELP-fusion partner
also influences T
t
. The hydrophobic tendamistat decreased
T
t
, whereas the hydrophilic thioredoxin slightly increased T
t
.
The authors speculated that interactions between the ELP
chain and solvent-exposed hydrophobic regions in tendami-
stat lead to the observed decrease in T
t
. However, biologic
activity of both target proteins as ELP fusion remained
unaffected [23]. Even though average yields of ELP-fusion
proteins were in the range of 10-100mg/L of E. coli culture
volume, optimization of culture conditions and medium, by
addition of frequent ELP-containing amino acids, boosted
the yield about 36-fold to the level of 1.6 g/L for GFP-ELP
[24]. Also, high cell density E. coli fermentation was
suitable for high-level production of ELP-fusion proteins
[25]. Recently, it has been shown that positioning the ELP
component at the C-terminus but not at the N-terminus of the
target protein can enhance expression levels, leading to an
improved yield of purified protein [26]. In the case of very
low ELP-fusion protein expression (defined as
100
m
g
soluble expressed protein per liter of culture medium),
the addition of excess free ELP mediates efficient phase
transition of ELP-fusion protein as the co-aggregation
between the ELP and the ELPylated target is highly specific
(Figure 14.2B) [27].
The ELP moiety can be cleaved from the target protein by
treatment with site-specific proteases such as factor X or
thrombin. The addition of proteases might interfere with the
structure of the target protein, such as minimal unspecific
cleavage of the protease within the ELP-fusion partner or
could raise cost issues for large-scale protein production. An
attractive source of proteases was recently developed by Lan
and co-workers. Here, the target protein and a protease were
ELPylated and purified from E. coli. After purification, the
ELP-target and the ELP-protease fusion proteins were
mixed, resulting in the cleavage of the target protein from
the ELP component. Finally, the target protein was separated
from ELP as well as the ELP-tagged protease by ITC (Figure
14.2C) [28].
The intein technology, designed to induce protein self-
cleavage, was combined with ELP technology to avoid cost
issues of postpurification protein cleavage [29]. Intein-ELP
fusions were designed to purify a number of native recom-
binant target proteins from E. coli [30,31]. This method
requires the introduction of an intein coding sequence
between the sequence encoding the target protein and the
ELP carrier protein resulting in the triple fusion “target
protein-intein-ELP”. First of all, the triple fusion is purified
by inverse transition cycling (ITC), and subsequently intein
self-cleavage is induced by pH/temperature shift or DTT
treatment to release the target protein from the intein-ELP
fragment. The intein-ELP carrier fragment could be then
efficiently removed by a second round of ITC because the
temperature-based precipitation step specifically affects the
ELP carrier protein (Figure 14.2D) [30,31]. For scale-up,
separation of aggregated ELP-fusion proteins could include
continuous centrifugation or tangential-flow microfiltration
[32]. The use of microfiltration including efficient prefiltra-
tion steps and the fine-tuned temperature and salt conditions
depending on the properties of the purified protein could
result in significant improvement of yield and purity [33].
High concentrations of sodium chloride, which are com-
monly used decreasing the T
t
are potentially corrosive,
thereby limits their use in industrial processes. Fong and
<
Search WWH ::
Custom Search