aggregation, high-swelling resins, technological advancements in
automated SPPS, and increased purity of the protected amino acids
have advanced the previously set limit for peptides of 50–70 amino
acids, depending on the sequence [3–6]. Still, the synthesis of
proteins, such as PDZ domains that comprise typically 80–100
amino acids, is generally not feasible by SPPS.
In conjunction with SPPS, the development of native chemical
ligation (NCL), enabled by the pioneering work of Kent and colleagues in 1994, expanded the range of protein size that can be
synthesized. This reaction involves a chemoselective conjugation of
one peptide bearing a C-terminal α-thioester with a second peptide
bearing an N-terminal Cys, resulting in a native peptide bond
[7]. Proceeding initially through transthioesterification driven by
nucleophilic attack of the N-terminal Cys thiol moiety to the carbonyl carbon of the thioester, a new intermolecular thioester intermediate is formed. Subsequently, this thioester intermediate
rearranges through an S-to-N acyl shift by the α-amine of the
Cys. This leads to the formation of a native amide bond (see
Fig. 1) [8, 9]. For the synthesis of larger proteins, ligation of several
peptide fragments is possible, either by stepwise ligation from the
N- to the C-terminus or by one-pot ligations with Cys protection
groups [10–13]. By applying NCL, several small proteins have been
successfully synthesized, such as the HIV-1 protease covalent
dimer, encompassing 203 amino acids, generated from four synthetic peptide fragments [14].
However, the synthesis of larger proteins (>20 kDa) by NCL
remains challenging, because the multiple ligation steps impact the
overall yield, and certainly cannot match the yields of recombinant
systems. Thus, to address these concerns, expressed protein ligation
(EPL), a derivative of NCL, was developed to leverage the advantages of both recombinant protein expression and SPPS. EPL
involves a reaction between a recombinant protein fragment with
a synthetic peptide fragment containing the desired chemical modification [8, 15]. Multistep ligations involving the joining of more
than two fragments can be performed, but the reaction always
requires a combination of synthetic and recombinant components.
Generally, there are two types of EPL strategies that can be used for
targeting the insertion of modifications at different positions of the
protein: (1) modifications in the N-terminal region, where a synthetic peptide is ligated to a recombinant fragment with an
N-terminal Cys, and (2) modifications in the C-terminal region
where an N-terminal recombinant thioester protein is joined with a
synthetic peptide.
In the first strategy, a recombinant protein fragment with an
N-terminal Cys residue is generated typically by introduction of a
cleavage site, such as a protease recognition site, immediately adjacent to the N-terminal Cys. Then, proteases such as Factor Xa or
enterokinase cleave and reveal the N-terminal Cys residue (see
Fig. 2). The peptide thioester can be generated by Fmoc- as well
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Christin Kossmann et al.
automated SPPS, and increased purity of the protected amino acids
have advanced the previously set limit for peptides of 50–70 amino
acids, depending on the sequence [3–6]. Still, the synthesis of
proteins, such as PDZ domains that comprise typically 80–100
amino acids, is generally not feasible by SPPS.
In conjunction with SPPS, the development of native chemical
ligation (NCL), enabled by the pioneering work of Kent and colleagues in 1994, expanded the range of protein size that can be
synthesized. This reaction involves a chemoselective conjugation of
one peptide bearing a C-terminal α-thioester with a second peptide
bearing an N-terminal Cys, resulting in a native peptide bond
[7]. Proceeding initially through transthioesterification driven by
nucleophilic attack of the N-terminal Cys thiol moiety to the carbonyl carbon of the thioester, a new intermolecular thioester intermediate is formed. Subsequently, this thioester intermediate
rearranges through an S-to-N acyl shift by the α-amine of the
Cys. This leads to the formation of a native amide bond (see
Fig. 1) [8, 9]. For the synthesis of larger proteins, ligation of several
peptide fragments is possible, either by stepwise ligation from the
N- to the C-terminus or by one-pot ligations with Cys protection
groups [10–13]. By applying NCL, several small proteins have been
successfully synthesized, such as the HIV-1 protease covalent
dimer, encompassing 203 amino acids, generated from four synthetic peptide fragments [14].
However, the synthesis of larger proteins (>20 kDa) by NCL
remains challenging, because the multiple ligation steps impact the
overall yield, and certainly cannot match the yields of recombinant
systems. Thus, to address these concerns, expressed protein ligation
(EPL), a derivative of NCL, was developed to leverage the advantages of both recombinant protein expression and SPPS. EPL
involves a reaction between a recombinant protein fragment with
a synthetic peptide fragment containing the desired chemical modification [8, 15]. Multistep ligations involving the joining of more
than two fragments can be performed, but the reaction always
requires a combination of synthetic and recombinant components.
Generally, there are two types of EPL strategies that can be used for
targeting the insertion of modifications at different positions of the
protein: (1) modifications in the N-terminal region, where a synthetic peptide is ligated to a recombinant fragment with an
N-terminal Cys, and (2) modifications in the C-terminal region
where an N-terminal recombinant thioester protein is joined with a
synthetic peptide.
In the first strategy, a recombinant protein fragment with an
N-terminal Cys residue is generated typically by introduction of a
cleavage site, such as a protease recognition site, immediately adjacent to the N-terminal Cys. Then, proteases such as Factor Xa or
enterokinase cleave and reveal the N-terminal Cys residue (see
Fig. 2). The peptide thioester can be generated by Fmoc- as well
194
Christin Kossmann et al.