Biomedical Engineering Reference
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discrepancy between the two research groups about the use
of large hydrophobic residues such as Phe, and large basic
and acidic residues, such as Arg and Glu, respectively.
Whilst George and Heringa [17] showed that these residues
were preferred in the linkers, Argos [18] suggested these
residues were generally excluded, because their hydropho-
bicity or bulky size could cause instability of the linker in
aqueous solution and affect the structural independency of
the linker.
amino acids. It also had a cyclic side chain structure and a
limited backbone angle for movement [21]. As a result,
linkers with a Pro-rich sequence might form relatively
rigid structures and avoid unwanted interactions with the
protein domains.
In summary, the natural linkers typically exhibited a
range of length between 4 and 20 amino acids. Several
amino acids (Pro, Thr, and Gln) were found to be favorable
linker components. The hydrophobicity of the natural link-
ers showed a tendency to decrease with the increase of linker
length. Various conformations were adopted by the natural
linkers, with
a
-helix and coil/bend secondary structures
found to be the most abundant. Longer linkers tended to
adopt more helical structures than shorter ones. Nonhelical
linkers appeared to incorporate high frequency of Pro to
increase the structural rigidity. Taken together, natural link-
ers exhibited extended conformations, good structural sta-
bility, and had no unfavorable interactions with the adjacent
protein domains. As a result, these oligopeptide linkers
could effectively separate the protein domains, allow inde-
pendent folding and functions of each domain, and reduce
the steric hindrance between them. The properties of natural
linkers could serve as a general guideline for the rational
design of linkers in fusion proteins.
4.2.3 Hydrophobicity of Linkers
The study by George and Heringa [17] also calculated the
average hydrophobicity of the linkers using Eisenberg's
normalized consensus residue hydrophobicity scale [19],
and assigned values ranging from 0 (hydrophilic) to 1
(hydrophobic) for each set of linker lengths. Small linkers
displayed a hydrophobicity value of 0.69
0.11, while large
linkers showed a lower hydrophobicity of 0.62
0.08. As
mentioned earlier, the linkers were more exposed in the
solvent with the increase of length. Here the data suggested
that longer linkers were more hydrophilic than smaller
linkers, and therefore, they could function more indepen-
dently in the solvent and allow the domains to move freely.
4.2.4 Secondary Structures of Linkers
4.3 EMPIRICAL LINKERS IN RECOMBINANT
FUSION PROTEINS
The linkers from natural proteins could adopt various con-
formations in secondary structure, such as helical,
b
-strand,
coil or bend, and turns, to provide their functions. Among
George and Heringa's database of linkers, 38.3% adopted
the
a
-helical secondary structure, 13.6% were in
b
-strands,
8.4% were in turns and the rest 37.6% were in coil or bend
secondary structures. The conformation preference of link-
ers also changed with their length. While small linkers
mainly adopted strand and coil secondary structures
(33.6% and 36.9%, respectively), large linkers were mainly
in helical and coil secondary structures (31.4% and 45.4%,
respectively). There was a tendency for linkers to adopt
more helical structures as their length became longer [17].
On the basis of their conformation, linkers could be
classified into two categories: helical and nonhelical. The
a
-helix was a well-designed and stable structure, with intra-
segment hydrogen bonds and a closely packed backbone
[20]. Therefore, many natural and empirical linkers (to be
discussed later) adopted this conformation. This type of
structure might act as a rigid spacer to separate protein
domains, facilitate their independent folding, and could
reduce unfavorable interactions between domains. On the
other hand, without an inherent rigid structure, the
nonhelical linkers tended to incorporate a high frequency
of Pro to fulfill the requirement for stiffness. As mentioned
previously, Pro had a cyclic side chain with no amide
hydrogen to form hydrogen bonding with any neighboring
On the basis of the criteria suggested by the natural linker
peptides, researchers have designed various empirical link-
ers for fusion proteins to achieve desirable functions. These
linkers can have various lengths, conformations, and com-
positions to fit into specific applications. Three types of
empirical linkers (flexible linkers, rigid linkers, and cleava-
ble linkers) are discussed in this section. Each category of
linkers has specific characteristics and usage in the construc-
tion of recombinant fusion proteins.
4.3.1 Flexible Linkers
Flexible linkers are generally composed of small amino
acids that can be nonpolar, such as Gly, or small, polar
amino acids, such as Ser or Thr. These linkers can maintain
structural stability in aqueous solutions by forming hydro-
gen bonds with the water molecules, and also provide good
mobility of the connecting functional domains. Flexible
linkers can be applied to join two functional domains
passively, while either allowing a certain amount of move-
ment of the protein domains, or facilitating the interaction
between domains.
4.3.1.1 Flexible Glycine and Serine Linkers The most
commonly used flexible linker peptides have sequences
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