Biomedical Engineering Reference
In-Depth Information
serum albumin (HSA) and interferon-
a
2b (IFN-
a
2b). Albu-
min fusion proteins are commonly used for prolonging the
plasma half-life of protein drugs and enhancing in vivo
efficacy [8,62,63]. Direct fusion of HSA and IFN-
a
2b
can cause interference between domains, and diminish the
antiviral activity of IFN-
a
2b domain [64]. Zhao et al.
compared the biological activity of HSA-IFN-
a
2b fusion
proteins with no linker or three types of linkers including
flexible linker (GGGGS), rigid linker (PAPAP), and helical
linker (AEAAAKEAAAKA) [11]. Previous studies from the
same group had indicated that the direct fusion of IFN-
a
2b
to HSA without a linker interfered with the disulfide bond
formation between two cysteine residues in the IFN-
a
2b
sequence. Their result from comparing the different types of
linkers showed that all the three linkers could separate HSA
and IFN-
a
2b effectively and restore the disulfide formation,
as all fusion proteins appeared as a homogenous product on
nonreducing SDS-PAGE. Compared to the fusion protein
without a linker, all the three linkers demonstrated a signifi-
cantly improved anti-viral activity of HSA-IFN-
a
2b by
39% (flexible linker), 68% (rigid linker), and 115% (helical
linker). These results suggest that structured linkers such as
the rigid and helical linkers may separate HSA and IFN-
a
2b
more effectively than flexible linker, as they increased the
anti-viral activity more significantly. The higher activity
associated with helical linker could not be simply ascribed
to its longer length, as increasing the length of flexible linker
from 5 amino acid residues (GGGGS) to 10 amino acid
residues (GGGGSGGGGS) could not increase the activity of
fusion protein.
These studies provide useful evidences that linker inser-
tion is an effective tool to improve biological activity of
fusion proteins. By inserting a flexible or helical peptide
linker that adopts a rigid structure, effective separation of
the two protein domains can be achieved. The unwanted
interaction and steric hindrance between the protein
domains could then be diminished, and, as a result, the
bioactivity may be greatly enhanced.
domains [64], the steric hindrance may not be effectively
reduced because of the short distance between domains.
Many linkers can provide longer distance between domains,
and allow for the independent folding of protein moieties.
Linker technology has been shown as a promising and
practical method to increase the expression yield of recom-
binant fusion proteins.
The effect of linker insertion on expression efficiency of
fusion proteins was observed in Tf-based fusion proteins
[12]. Tf was first directly fused with hGH to construct a
fusion protein for Tf receptor-mediated oral delivery of
hGH. Fusion proteins with two orientations of Tf and
hGH (hGH-Tf and Tf-hGH) were constructed to achieve
the optimal bioactivity. A helical (H4)
2
linker (A(EAAA-
K)
4
ALEA(EAAAK)
4
A) was inserted in the two fusion
proteins between hGH and Tf since this linker may improve
the bioactivity of the fusion proteins [14]. The resultant
fusion proteins were designated as hGH-(H4)
2
-Tf and
Tf-(H4)
2
-hGH. These fusion proteins were then transiently
expressed from HEK293 cells. Surprisingly, the insertion of
the helical linker (H4)
2
greatly improved the expression
level of the Tf-fusion proteins. The insertion of the helical
linker in hGH-Tf fusion proteins resulted in a 1.66-fold
higher expression in HEK293 cells. Similarly, fusion protein
Tf-(H4)
2
-hGH with the reversed orientation had an expres-
sion level 2.39-fold higher than that of Tf-hGH.
A more profound effect of linker insertion on protein
production was observed in Tf fusion proteins that were
fused to G-CSF. Two fusion proteins with reverse orientation
(G-CSF-Tf and Tf-G-CSF) were first constructed. The heli-
cal linker (H4)
2
was then inserted into G-CSF-Tf to test if it
increases the expression level. A moderate enhancement
(1.44-fold) was obtained for G-CSF-(H4)
2
-Tf compared to
G-CSF-Tf. Unexpectedly, the Tf-G-CSF fusion protein,
which had the reversed orientation, failed to be efficiently
expressed fromHEK293 cells, with its quantity too low to be
detected convincingly by Western blot analysis. Even with
the insertion of a flexible linker (GGGGS)
3
, the expression
of the Tf-(GGGGS)
3
-G-CSF fusion protein was still
unsuccessful. Dramatically, when the helical linker (H4)
2
was inserted, high level of Tf-(H4)
2
-G-CSF was expressed
from the cells, with an 11.2-fold elevation on production.
The effect on the expression level did not simply depend on
the length of the linker, because Tf-G-CSF with a linker that
was encoded with the reversed coding sequence for the
(H4)
2
(which produces an identical peptide length without
a helical structure) failed to express.
As suggested by these studies, when difficulties are
encountered for fusion protein production, a feasible
approach is to insert linkers between functional domains.
The exact mechanism for the improved expression with
helical linkers is still unclear. The rigid, extended nature
of the helical linker may effectively separate the two protein
domains, and greatly reduce the interference between them.
4.4.2 Linkers Can Increase Expression Yield
of Fusion Proteins
Gene fusion techniques offer the advantage to combine the
desired properties from different proteins into a fusion
protein. However, direct fusion of two protein moieties
may cause folding defects resulting from structural pertur-
bation between different moieties. Many researchers have
reported the difficulties in expressing stable and homoge-
nous fusion proteins [11,14]. The instabilities of fusion
proteins often result in a low expression yield and a hetero-
geneous product, which are both unfavorable characteristics
for downstream applications. Although the expression level
of fusion proteins can sometimes be improved by simply
switching the orientation of the two component protein
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