Biomedical Engineering Reference
In-Depth Information
peptides extending from the carboxyl terminus of the V
L
to
the amino terminus of the V
H
. The linker contained several
glycine and serine residues for flexibility, as well as glutamic
acid and lysine residues to improve the solubility.
Another application of flexible linkers is in the produc-
tion of tagged fusion proteins. Various tags, such as a
Myc-tag (10 amino acid residues) derived from the c-myc
gene product, glutathione S-transferase (GST)-tag (220
amino acid residues), and histidine (His)-tag (
the protein G domain in a protein G-Vargula luciferase
fusion protein is not retained with the insertion of a flexible
GGGGS linker [33]. In this case, the insertion of a flexible
linker may not be sufficient to separate the functional
domains and prevent the unfavorable interactions between
the domains. Therefore, for applications where the preven-
tion of interdomain interference is necessary for activity, a
rigid linker that has a stiff structure should be applied to
effectively separate the domains and allow them to act
independently.
6 histidine
amino acid residues), can be fused to proteins of interest for
improving expression, and for facilitating purification or
identification. The direct fusion between a protein and a tag,
however, may cause the loss of function because of the
interference from the tag sequence. In order to overcome this
drawback, Sabourin et al. reported the construction of
tagged fusion proteins using a flexible linker, (Gly)
8
[31].
When the flexible (Gly)
8
linker was inserted between a Myc
epitope tag and the protein of interest, Est2p, the function-
ality of epitope-tagged Est2p was greatly improved com-
pared to the fusion protein without a linker. The positive
effects of the flexible protein linkers were likely due to the
proper folding of epitope-tagged proteins after linker inser-
tion, as well as the reduced steric hindrance between func-
tional domains.
In another study, a flexible linker, GSAGSAAGSGEF, was
described byWaldo et al. [32]. This linker sequence excluded
large bulky hydrophobic residues as suggested by Argos [18].
The GSAGSAAGSGEF linker was initially designed to con-
struct GFP-fusion proteins for rapid protein-folding assay.
This assay was developed to detect the correct folding of a
protein by expressing a protein of interest as an N-terminal
fusionwithGFP, and has since been applied to numerous other
fusion proteins. This linker provided similar performance of
the GFP folding reporter as a longer (GGGS)
4
linker. It was
chosen over the (GGGS)
4
linker because it reduced the
amount of homologous repeats in the DNA coding sequence,
which could result in deletions by homologous recombination
during the shuffling protocol for cloning.
In summary, flexible linkers are mainly composed of
small or polar amino acids such as Gly and Ser. These linkers
can provide distance between functional domains while
allowing the necessary interactions or movement of the
functional domains. By adjusting the copy number of the
linkers, the optimal length of the linkers can be identified to
achieve proper folding, good production, or the highest
biological activity.
4.3.2.1 Alpha Helix-Forming Linker (EAAAK)
n
A
new type of rigid linkers, the
a
helix-forming peptides
A(EAAAK)
n
A(n
2-5) were introduced for the construc-
tion of bioactive fusion proteins by Arai et al. [34,35]. These
linkers were shown to adopt an
a
-helix conformation, which
was stabilized by a Glu
-Lys
þ
salt bridge within the linker
structure [36].
These helical linkers were first characterized by inserting
various sequence lengths between enhanced blue fluorescent
protein (EBFP) and enhanced green fluorescent protein
(EGFP), and measuring the fluorescent resonance energy
transfer (FRET) efficiency [34]. FRET is a distance-dependent
interaction, where a shorter distance between the two fluo-
rophores (i.e., EBFP and EGFP) will result in a higher FRET
efficiency. In this study, the FRET efficiency between the two
domains decreased as the length of helical peptides increased,
indicating that these linkers can control the distance between
domains by changing repetitions of the EAAAK motif. Com-
pared to the flexible linkers with the same number of amino
acid residues, the helical linkers induced much less FRET
efficiency when inserted into EBFP-EGFP fusion proteins.
This result suggested the distance between protein domains
did not simply depend on the number of residues in the linker,
but could also be affected by the conformation of the linker.
The circular dichroism (CD) spectroscopic analysis of the
fusion proteins confirmed the
a
-helical conformation of
the linkers, and indicated that the helix content increased
as the length of the linker increased [34].
Another study by Werner et al. [37] utilized helical linker
technology to develop a virus-protein A system to efficiently
manufacture protein A-based immunosorbent nanoparticles
for use in industrial purification of monoclonal antibodies.
This report showed that a functional fragment of protein A
(133 aa) could be displayed on the surface of a tobamovirus
as a C-terminal fusion to the viral coat protein via a 15-aa
linker (EAAAK)
3
[37]. Without a linker, only peptides
shorter than 20 aa could be fused to the coat protein of
plant rod-shape viruses without preventing the assembly of
functional virions [38]. In addition, in the case of tobamo-
virus, viral particles could form only when excess free coat
protein was co-expressed with the fusion protein, leading to
the formation of viral particles displaying the protein A
fragment on only 5% of
¼
4.3.2 Rigid Linkers
Although the flexible linkers are versatile for constructing a
wide variety of chimeric fusion proteins, it has limitations
such as ineffectiveness in spatially separating functional
domains due to its high flexibility. For instance, Maeda
et al. reported that the immunoglobulin binding ability of
the coat protein subunits.
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