Biomedical Engineering Reference
In-Depth Information
Dramatically, with the help of a 15-aa helical linker between
the coat protein and the protein A fragment, the fusion
protein could be successfully expressed and assembled
into functional virions in the absence of free coat proteins.
This example clearly demonstrated the pivotal role of linker
insertion to provide proper distance between functional
domains and to allow for particle assembly.
Lu et al. used the
a
-helical peptides (EAAAK)
n
(n
interferon (IFN)-
g
-gp120 fusion protein [44]. With the short
10-aa linker, the fusion protein possessed a relatively low
biological activity of IFN-
g
. By gradually increasing the
length of the Ala-Pro linker, the bioactivity had been
improved. The chimeric protein containing the longest
34-residue linker had the highest activity, measured at
88% of that of the control protein. In comparison, fusion
proteins with cysteine-tryptophan linkers, constructed by
inserting the oligonucleotides encoding linker in the reverse
orientation, possessed no detectable biological activity,
indicating that the improvement of the bioactivity by the
(Ala-Pro)
n
linker is sequence specific.
1-3)
as linkers to construct bifunctional fusions of
b
-glucanase
and xylanase enzymes [3]. The results showed that insertion
of helical linkers improved the catalytic efficiencies of both
moieties. The
b
-glucanase thermal stabilities of the fusion
proteins also improved with the increased length of the
a
-helical linkers. More interestingly, all of the fusion pro-
teins with helical linkers were more thermally stable than
those with the flexible linkers. Similar to the previous
studies, these trends were attributed to the rigid structure
of the
a
-helical linker that might decrease interference
between the linked moieties, suggesting that changes in
linker structure and length could affect the stability and
bioactivity of functional moieties.
ΒΌ
4.3.3
In Vivo
Cleavable Linkers
The linkers in fusion proteins discussed in this chapter so far
generally consist of stable peptide sequences, and can
provide structure flexibility, improve protein production
and stability, or increase biological activity. However, stable
peptide linkers do not allow for the separation of the two
fusion protein domains in vivo, and can have several draw-
backs including decreased bioactivity, steric hindrance
between functional domains, and altered biodistribution
and metabolism of the protein moieties because of interfer-
ence with each other.
4.3.2.2 Rigid (XP)
n
Linkers Another type of rigid link-
ers are composed of a proline rich sequence, (XP)
n
, where X
can be any amino acid, among which the common choices
are Ala, Lys, and Glu. Pro is a very unique amino acid, with
its side chain cyclized back to the amide on the backbone. As
suggested by George and Heringa [17], Pro has no amide
hydrogen to form hydrogen bond with other amino acids,
and therefore, it structurally prevents the interaction
between the linkers and the protein domains. Another
characteristic of Pro is that it has a very restricted confirma-
tion, with the backbone angle
4.3.3.1
In Vivo
Cleavable Disulfide Linker To over-
come some of these potential pitfalls in stable peptide
linkers, Chen et al. designed an in vivo cleavable linker
for recombinant fusion proteins utilizing the reversible
nature of the disulfide bond [45]. The reversible disulfide
linkage has been widely applied in drug delivery by chemi-
cal conjugation methods. However, the major pitfall in
chemically conjugating protein moieties through disulfide
bond formation is the generation of a range of heterogeneous
products. This novel recombinant fusion protein approach
described by Chen et al. offers the significant advantage in
the generation of a precisely constructed, homogeneous
product.
The cleavable dithiocyclopeptide linker contains a
thrombin-sensitive sequence, as well as an intramolecular
disulfide bond formed between two cysteine residues of the
linker (Figure 4.1). This linker was inserted between G-CSF
and Tf to construct a recombinant fusion protein. Treatment
in vitro with thrombin results in cleavage of the thrombin-
sensitive sequence, while the reversible disulfide linkage
between the two domains of the fusion protein remains.
After successful construction of the cleavable linker into
G-CSF-Tf fusion protein (designated as G-C-T), the in vitro
G-CSF biological activity of the fusion protein was deter-
mined via cell proliferation assay. The fusion protein was
pretreated with thrombin and reduced by DTT to mimic the
expected in vivo released free G-CSF. With the release of
free G-CSF, the fusion protein exhibited a two fold increase
limited to a small range of
w
65
. Having Pro resides in nonhelical linkers can increase
the stiffness, and allows for effective separation of the
protein domains. As a result, this type of linker usually
adopts rigid and extended structure. The (XP)
n
linker occurs
naturally in the E2 chain of the pyruvate dehydrogenase [39]
and in the IIABMan subunit of the Escherichia coli mannose
transporter [40].
The structure of this type of linker was extensively
studied by several groups [39,41,42]. For example, the
conformation of the (Ala-Pro)
7
dipeptide repeat in the alkali
light chain of skeletal muscle (LC1) was studied by
Bhandari et al. by using
1
H-NMR spectroscopy [41].
They concluded that this peptide linker adopted an extended
and rigid conformation because of the conformational
restrains imposed by the Pro, which strongly preferred
w
65
[43]. A similar study of
33-residue peptides containing repeating -Glu-Pro- or -Lys-
Pro- by Evans et al. also demonstrated the X-Pro backbone
had a relatively stiff elongated conformation [42].
The effect of sequence length in the (Ala-Pro)
n
peptide
linker (10-34 aa) was evaluated in a study using an
backbone angles close to
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