Biomedical Engineering Reference
In-Depth Information
4.3.3 P
EPTIDE
/P
ROTEIN
L
IGATION
A conceptually different strategy for the modii cation of proteins is to employ methods based on
solid-phase peptide synthesis (SPPS) for the generation of proteins. This would allow the incorpora-
tion of principally any amino acid, and thus circumvent the problems of incorporating d-amino acids,
which is not feasible by unnatural mutagenesis. SPPS has in a few cases been applied for the synthesis
of proteins, although yields are generally rather low. The i rst example was the synthesis of ribonuclease
A (124 residues) by Bruce Merrii eld in 1966 and since then a few other proteins have been prepared
by this approach, most notably HIV protease (99 residues), which enabled structural characteriza-
tion of the protein with inhibitors bound.
However, SPPS is generally limited to the preparation of up to 40-60 amino acid peptides,
whereas most proteins are considerably larger. Therefore, there has been a considerable interest in
developing methods that are not coni ned to these restrictions and in 1994, a strategy for the prepa-
ration of proteins from peptide fragments was introduced, called native chemical ligation (NCL,
Figure 4.10). In NCL, two or more unprotected peptide fragments can be ligated together, generat-
ing a (native) cysteine residue in the ligation site. The ligation requires a peptide with a C-terminal
protein thioester and a peptide with an N-terminal cysteine residue: the thiolate of the N-terminal
cysteine attacks the C-terminal thioester to affect transthioesterii cation, followed by the formation
of an amide bond after S ®
N
acyl transfer (Figure 4.10). The reaction takes place in aqueous buffer
and generally proceeds in good to excellent yield.
Thus, NCL is a very useful approach for the total chemical synthesis of proteins and has been
used for the preparation of numerous proteins, including glycoproteins and proteins with l uorescent
labels. An example is the synthesis of an analog of erythropoietin (EPO), which was derivatized with
monodisperse polymer moieties in order to improve the duration of action
in vivo
. The 166-residue
O
SN
acyl shift
O
O
transthioesteriication
H
2
N
O
Peptide 2
N
HN
O
SR
Peptide 1
Peptide 1
Peptide 2
Peptide 2
S
Peptide 1
O
HS
SH
(a)
O
HN
O
Peptide
H
2
N
Peptide
O
O
O
HS
N
Protein
S
Peptide
Protein
Protein
SR
O
SH
O
Protein
S
H
2
N
Intein
HS
O
Protein
H
Intein
(b)
FIGURE 4.10
Principles of NCL and EPL. (a) NCL: a peptide with an N-terminal cysteine and another
peptide with a C-terminal thioester can be ligated together. Initially, a reversible transthioesterii cation takes
place and subsequently
S
®
N
acyl shift, leading to a cysteine in the ligation site. (b) EPL is applying the same
principles, but one of the reactants is a recombinantly expressed protein, which allows the semisynthesis of
larger proteins.
