FUSION PROTEIN LINKERS: EFFECTS ON PRODUCTION, BIOACTIVITY, AND PHARMACOKINETICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 58

consisting primarily of stretches of Gly and Ser residues
(“GS” linker). These amino acids are suitable for flexible
linker peptides because the polar Ser amino acids allow
hydrogen bonding with the solvent, and the small size of Gly
and Ser provide the necessary flexibility [18]. The most
widely used example of a flexible linker has the sequence of
(Gly-Gly-Gly-Gly-Ser) n . By adjusting the copy number “n,”
the length of this GS linker can be fine tuned to achieve
suitable separation of the adjacent domains, or to allow for
the necessary interaction between domains.
The first notable application of the GS linker is the
construction of single-chain variable fragment (scFv), an
antigen-binding fusion protein composed of antibody light-
chain variable regions (V L ) tethered to heavy chain variable
region (V H ) via an oligopeptide linker [22]. By joining via a
peptide linker, the V H and the V L domains can be synthe-
sized as a single polypeptide chain to obtain equal molar
ratios, while the covalent linkage between V H and V L
facilitates the association of the two domains. An optimal
linker for scFv should adopt the proper dimension to bridge
the distance between the two domains, and exhibit an
independent secondary structure that will not

fusion proteins could bind to the Tf receptors expressed on
intestinal epithelium cells, and were efficiently transcytosed
across the intestinal absorption barrier. This approach was
widely applied for the oral delivery of various protein and
peptide drugs and exhibited marked pharmacological effects
[26,27]. For the G-CSF-Tf fusion protein, the in vitro
bioassay revealed a retention of less than 10% bioactivity
by the fusion protein compared to the parent free G-CSF.
The interference between the two domains most likely
decreased the binding of the fusion protein to G-CSF
receptors and might account for the suboptimal in vitro
and in vivo activities of the fusion protein. In order to
optimize the intrinsic bioactivity of the fusion protein, a
flexible GS linker, (GGGGS) 3 , was inserted to keep the two
domains at a distance [14]. The resultant fusion protein
displayed a profound increase of bioactivity, with an EC 50 of
0.20 ng/mL, about 10-fold less than that of the fusion protein
without a linker (EC 50 ¼ 2.34 ng/mL). This improvement

might be due to the reduced blockage of receptor binding
sites on G-CSF with a longer distance to the Tf domain.
In another study, the (GGGGS) 3 linker was inserted
between two copies of HBsAg preS1 (21-47) peptides to
produce a fusion protein for anti-preS1 (21-47) antibody
detection [28]. With the linker insertion, the immunoactivity
of the fusion protein is much stronger compared to the fusion
protein without a linker. This flexible linker allowed more
antibodies to bind to the fusion protein probably because of
the increased distance between the two domains and their
greater freedom of movement.
Another interesting example of the (GGGGS) n linkers
was the construction of self-renaturing enzymes. Enzyme-
chaperone chimeric protein was constructed as a new
approach to stabilize an enzyme [29]. Three soluble chi-
meric proteins were constructed, each consisting of a fusion
of chaperon domain (rTHS) and the enzyme of interest
(model penicillin amidase, PGA) separated by a flexible
(GGGGS) n linker with different copy numbers (n

interfere

with the protein folding of either domain.
Huston et al. selected (GGGGS) 3 to connect the carboxyl
terminus of the V H to the amino terminus of the V L [23]. This
linker was chosen because it has no ordered secondary
structure or tendency to interfere with the folding of the
protein domains. As a result, the two variable domains could
assume a natural orientation and conformation for antigen
binding. The length of the linkerwas also taken into account to
allow for the correct conformation of the scFv. The distance
between the C terminus of the V H domain and the N terminus
of the V L domain was about 3.5 nm according to the X-ray
data. Three copies of the GGGGS linker were selected since
its length, calculated to be about 5.7 nm, is able to bridge the
distance between the V H and V L domains.
Interestingly, the length of the linkers between the V H and
V L domains of a scFv could determine the association status
of the fusion protein. By shortening the length of the linker
to less than 12 residues, the V H and V L domains of a single
scFv could no longer pair with each other. Instead, two scFv
fusion proteins formed a dimer by associating the V H
domain from one fusion protein with the V L from another.
The resultant bivalent scFv dimer was termed diabody [24].
Furthermore, when the length of the linker was shortened to
less than 3 residues, the distance between V H and V L was
only enough to allow for the formation of a trimer, termed
triabody [25].
Besides scFv, the flexible linkers (GGGGS) n were also
widely applied in the construction of many other recombi-
nant fusion proteins. For example, a direct fusion of granu-
locyte colony-stimulating factor (G-CSF) and transferrin
(Tf) was constructed by Bai et al. for oral delivery of
G-CSF via Tf receptor-mediated transcytosis [13]. The Tf

1, 2, or 4).
The bioactivity of the chimeric protein improved as the length
of the linker increased. The catalytic activity of the chimeric
proteinwith the longest linker was almost equivalent to that of
the parent enzyme PGA. This study indicated that by adjust-
ing the linker length, the unfavorable inter-domain interfer-
ence could be minimized, and the bioactivity of the fusion
protein could achieve optimal conditions.

¼

4.3.1.2 Other Flexible Linkers Besides the (GGGGS) n
linker, other flexible linkers have been applied in construct-
ing recombinant fusion proteins. These linkers are also rich
in G and S residues in order to maintain their flexibility.
Bird et al. [30] selected the flexible linker sequence
(EGKSSGSGSESKST) to fuse the V H and V L domains of
immunoglobulin and constructed a bioactive scFv antibody.
Using computational methods and computer graphics, this
linker was designed by adding single amino acids or short