REVIEW ARTICLE
Expressed protein ligation
Method and applications
Ralf David, Michael P.O. Richter and Annette G. Beck-Sickinger
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany
The introduction of noncanonical amino acids and bio-
physical probes into peptides and proteins, and total or
segmental isotopic labelling has the potential to greatly aid
the determination of protein structure, function and protein–
protein interactions. To obtain a peptide as large as possible
by solid-phase peptide synthesis, native chemical ligation
was introduced to enable synthesis of proteins of up to 120
amino acids in length. After the discovery of inteins, with
their self-splicing properties and their application in protein
synthesis, the semisynthetic methodology, expressed protein
ligation, was developed to circumvent size limitation prob-
lems. Today, diverse expression vectors are available that
allow the production of N- and C-terminal fragments that
are needed for ligation to produce large amounts and high
purity protein(s) (protein a-thioesters and peptides or pro-
teins with N-terminal Cys). Unfortunately, expressed pro-
tein ligation is still limited mainly by the requirement of a Cys
residue. Of course, additional Cys residues can be introduced
into the sequence by site directed mutagenesis or synthesis,
however, those mutations may disturb protein structure
and function. Recently, alternative ligation approaches have
been developed that do not require Cys residues. Accord-
ingly, it is theoretically possible to obtain each modified
protein using ligation strategies.
Keywords: expressed protein ligation; IMPACT
TM

N-terminal Cys activated with an S-(methoxycarbonyl)sul-
fenyl (Scm) group of a second peptide. The free amino
function of this amino acid can attack the carbonyl group of
theesterandanOfiN-acyl transfer results in an amide-
bond. Reductive cleavage of the disulfide releases the free
Cys sidechain [8]. CNBr-cleavage fragments refold and
form noncovalent complexes and finally the missing peptide
bonds are reattached [9]. Cytochrome c CNBr fragments
1–65 and 66–104 were modified and religated by this
method [10], but this technique is limited by the occurrence
of Met at the cleavage site.
Dawson et al. introduced a simple and elegant method
called native chemical ligation (NCL) for the synthesis of
peptides by condensation of their unprotected segments.
The coupling of synthetic peptide-thioesters with peptides
carrying an N-terminal Cys leads to an amide-bond at the
ligation site. This approach has proven to be useful for
the synthesis of smaller proteins up to 120 amino acids in
Correspondence to A. G. Beck-Sickinger, Institute of Biochemistry,
University of Leipzig, Bru
¨
derstr. 34, D-04103 Leipzig, Germany.
Fax: + 49 341 97 36 909, Tel.: + 49 341 97 36 900,
E-mail:
Abbreviations: BAL, backbone amide linker; CBD, chitin binding
domain; eGFP, enhanced green fluorescent protein; EPL, expressed
protein ligation; FRET, fluorescence resonance energy transfer;
GFP, green fluorescent protein; HOBt, 1-hydroxybenzotriazole;
IMPACT
TM

thioesterification step is reversible and no side products
are obtained, thus, no protecting groups are necessary. An
alternative method was introduced by Tam et al. [16,17],
where a C-terminal thiocarboxylic acid S-alkylates an
N-terminal a-bromoAla to form a covalent thioester. This
rearranges by SfiN-acyl shift and builds an -X-Cys- peptide
bond (Fig. 1).
To prevent the thiol of the N-terminal Cys from oxidation,
and thus forming an unreactive disulfide linked dimer, it is
necessary to add thiols or other reducing reagents like tris(2-
carboxyethyl)phosphine (TCEP) [18] to the reaction mix-
ture. Furthermore, the addition of an excess of thiols not
only keeps the thiol-functions reduced but also increases the
reactivity by forming new thioesters through transthioeste-
rification [19]. The addition of solubilizing agents such as
urea or guanidinium hydrochloride does not affect the
ligation reaction and can be used to increase the concentra-
tion of peptide segments and results in higher yields. The
compatibility and efficiency of all proteinogenic amino acids
at the C-terminus of the thioester peptide to react in NCL
was determined by Hackeng et al.[20].All20aminoacids
except Val, Ile and Pro can be placed in the -X-Cys- position
in NCL. Val, Ile and Pro are reported to react slowly. Also,
Asp and Glu as C-terminal residues are less favourable
because of the formation of side products [21].
A useful application of NCL is solid-phase chemical
ligation (SPCL) [22]. In this approach, one of the two
segments is bound to a polymer, while the other is applied in
aqueous solution and can be used inexcess. A simple washing
step completely removes the solubilized peptides and the

amide Ôsafety catchÕ linker [30]. The C-terminus of the
growingpeptidechainisattachedtotheresinwithanacid-
and base-stable N-acyl sulfonamide linker. The sulfonamide
is activated after peptide synthesis by N-alkylation using
diazomethane or iodoacetonitrile. The cleavage occurs with
nucleophile like thiols, which finally results in a peptide
thioester [31,32]. In the backbone amide linker (BAL)
strategy, the first carboxy terminally protected amino acid is
attached to the resin on the backbone nitrogen. The peptide
chain grows in the N-terminal direction. Deprotection,
activation and thioester formation at the carboxy terminus
occurs on the solid support. The peptide thioester can be
cleaved from the resin with trifluoroacetic acid [33].
Another approach uses standard resins like phenyl-
acetamidomethyl (PAM) or 4-hydroxymethyl benzoic acid
(HMBA), the Lewis acid, Al(CH
3
)
2
Cl and thiols in
Fig. 1. Ligation of unprotected peptide segments. In native chemical
ligation (A) the first step is a transthioesterification of a Ca-thioester by
the thiol function of an N-terminal Cys followed by a spontaneous
SfiN-acyl shift to obtain a native peptide bond. In an alternative
approach (B), a Ca-thiocarboxylic acid reacts with an a-bromo amino
acid by forming a thioester. This leads to the same product as in
method A.
664 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004
methylenchloride [34]. Unfortunately, the alkylaluminium
thiolate method can lead to epimerization at the C-terminus

clear. However, understanding of inteins, their evolution,
distributions and properties, will be easier if they are
considered as parasitic genetic elements. They will not
contribute to an organism’s fitness if they are propagated
into the next generation. The insertion of an intein gene into
a protein gene can be described through the so called
homing cycle. Homing is the transfer of a parasitic genetic
element to a cognate allele that lacks the element. This
process results in the duplication of the parasitic genetic
element and its rapid spread in a population [41–43]. Inteins
occur in organisms of all three domains of life as well as in
viral and phage proteins. There they are predominantly
found in enzymes involved in DNA replication and repair
[40,44]. Inteins can be divided into four classes: the maxi
inteins (with integrated endonuclease domain), mini inteins
(lacking the endonuclease domain), trans-splicing inteins
(where the splicing junctions are not covalently linked) and
Ala inteins (Ala as the N-terminal amino acid) [45]. The
sequences of inteins have some characteristics in common.
They appear in conserved regions of the host protein and all
intein sequences harbour different motifs termed A and B
(which contain a conserved Thr and His) at the N-terminal
splicing domain, F and G at the C-terminal splicing domain
(Fig. 3). Endonuclease containing inteins also bear the
blocks C, D, E and H [38,46]. The N-terminal amino acids
are typically Cys, Ser or Ala. The C-terminal block G
contains a conserved His/Asp pair and a downstream Cys,
Ser or Thr amino acid.
The nucleophilic thiol or hydroxyl sidechains of the
conserved amino acid residues led to the assumption that

intein [47,48].
Splicing mechanism
The first step of the well understood standard splicing
process of inteins (Fig. 4) is the transfer of the N-terminal
extein unit to the sidechain -SH or -OH group of a Cys/Ser
residue located at the immediate N-terminus of the intein
(NfiS-acyl shift). In some cases, inteins bear Ala at the
ultimate position at their N-terminus. In such cases, the first
step is circumvented [48,49] and the +1 nucleophile within
the C-extein attacks the carbon of the peptide’s N-terminal
splicing junction. This rearrangement seems to be thermo-
dynamically highly unfavourable but the molecular archi-
tecture of the intein forces the scissile peptide bond into a
twisted conformation of higher energy and thereby pushes
the equilibrium to the (thio)ester side. The following step is a
new transfer of the N-terminal extein to the Cys/Ser/Thr at
the +1 position of the C-extein, which leads to a branched
intermediate. In the last step, which might be a concerted
reaction, a conserved Asp residue at the C-terminus of the
intein cyclizes and a peptide bond is formed between the two
exteins through an SfiN-acyl shift [50].
This splicing mechanism implicates the importance of the
conserved amino acids flanking the splicing junctions such
as the block B Thr and His, and the block G His [45].
In the case of C-terminal splicing, the cumulative data
indicate that the present penultimate His appears to assist
the C-terminal Asp cyclization, although there are reported
mutants referring to this residue which did not prevent
splicing. The three dimensional structure of the splicing
domain at the N-terminal part of the intein forces the

acyl shift and yield a peptide bond. Peptide
bond cleavage can occur independently at
both splicing sites. Mutation of Cys
1
to Ala
prevents splicing at the N-terminus and leads
to a C-terminal extein bonded with the intein.
C-terminal splicing cannot occur when the
C-terminal Asn is substituted by an Ala
residue and the N-terminal extein is cleaved
by nucleophilic attack.
666 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004
acids. Both play a key role for the N-terminal splicing
process. Substitution of block B His to Leu in Sce VMA
abolished splicing [53,54] and only C-terminal cleavage
occurred. This implies that this His residue takes part in the
first NfiS rearrangement at the N-terminal splicing junc-
tion. X-ray crystal structures of Sce VMA1 [55–57] and
Mxe GyrA [51] with exteins showed a protonation of the
scissile peptide bond through the imidazole ring. This
interaction promotes the breakdown of the tetrahedral
intermediate formed by the +1 nucleophilic attack of the
N-terminal thioester bond. These findings were further
elucidated and confirmed through investigations of Ala
inteins. The exact role of Thr is not yet fully understood
because of the lack of available structural data. It has been
postulated that the Mxe GyrA intein stabilizes the tetra-
hedral intermediate at the N-terminal splicing junction by
the formation of an oxy anion hole through Nd of Asn74
and the block B Thr.

activated proteins, which can further be ligated with peptide
segments and provides access to artificially labelled proteins;
(d) inteins facilitate the synthesis of cyclic proteins and
(e) inteins are used for the detection of protein–protein
interactions [45,46].
Three dimensional structures of inteins
The structure of the intein Sce VMA1 that was determined
by X-ray crystallography clearly shows two domains
(Fig. 5) [55–57]. The structure of the splicing domain is
similar to that of the mini intein in the Mycobacterium
xenopi gyrase (Mxe GyrA) [51]. Residues from the endo-
nuclease domain of Sce VMA1 contribute to target
sequence-specific contacts as well as parts of the other
domain that are distant from the Sce VMA1 cleavage site.
Several studies have been made by photo-crosslinking to
identify these residues [60]. The splicing domains have
predominantly all b-structures and show high similarity to
the structure of the hedgehog proteins that are important in
the development of multicellular organisms [61].
Formation of C-terminal thioester-activated proteins
Protein engineering via NCL requires the specific generation
of C-terminal thioester-tagged proteins allowing ligation
with a second peptide or protein containing an N-terminal
Cys or Ser residue. The potent synthesis of Ca-thioesters of
bacterially expressed proteins was found through studies of
the N-terminal cleavage mechanism of inteins. In general,
the cleavage of the peptide bonds at either the N-terminus or
the C-terminus of the intein can occur independently.
Replacement of the C-terminal Asp by Ala blocked the
splicing process in the Pyrrococcus species GB-D intein.

with an affinity chitin binding tag (Fig. 6)] is commercially
available from New England Biolabs and allows the single
column isolation of protein thioesters by utilizing the thiol
induced self-cleavage activity of various inteins. In this
system, the target gene is cloned into an expression vector
right at the N-terminus of a modified intein. An additional
chitin binding domain (CBD) from Bacillus circulans is
fused to the C-terminal part of the intein and enables the
affinity purification of the further expressed three segmental
fusion proteins. All other cell proteins can be washed away
from the absorbed fusion protein, and after induction of the
cleavage with an excess of thiol and overnight incubation,
the protein of interest can be eluted as a C-terminal thioester
from the chitin resin. Several inteins are available (Table 1)
which differ with respect to the thiols used at 4 °C.
Additionally, there are recombinant inteins, which cleave
the C-terminal extein through the change of the pH or
temperature. This can be applied to protein purification or
EPL for the synthesis of the Cys segment. In the case of
C-terminal thioester synthesis, modified mini inteins are
commonlyusedwithaAsnfiAla mutation from the genes
of Mycobacterium xenopi (Mxe GyrA), Saccharomyces
cerevisiae (Sce VMA), Methanobacterium thermo-autotro-
phicum (Mth RIR1) and Synechocystis sp. PCC6803 (Ssp
DnaB). The cleavage takes place only at the N-terminus of
the intein because of the absence of the Asp cyclization.
These inteins can be cleaved through induction with various
thiols in great efficiency. This is an important chemical
aspect for ongoing protein ligation together with the
thioester stability.

(CBD). Both fragments can be synthesized by SPPS and specifically
labelled at the N- or C-terminus of the protein. Ligation of both
fragments proceeds under the conditions of NCL.
Table 1. Intein based vectors and their potential applications. Mxe GyrA, Mycobacterium xenopi gyrease A; Mth RIR1, Methanobacterium ther-
moautotrophicum; Ssp DnaB, Synechocystis sp. PCC6803; Sce VMA, Saccharomyces cerevisiae.
Vector Intein Splice junction Cleavage induction References Applications
pTXB1, 3 Mxe GyrA C-terminus Thiol
a
[64] Purification, generation of C-terminal thioesters
pTYB1, 2 Sce VMA C-terminus Thiol
a
[62] Purification, generation of C-terminal thioesters
pTWIN1 Ssp DnaB N-terminus pH and temperature [88] Purification C-terminal thioesters, aCys-proteins,
protein ligation, cyclization
Mxe GyrA C-terminus Thiol
a
[88]
pTWIN2 Ssp DnaB N-terminus pH and temperature [111] Purification, C-terminal thioesters, aCys-proteins,
protein ligation, cyclization
Mth RIR1 C-terminus Thiol
a
pTYB11, 12 Sce VMA N-terminus Thiol
a
[112] Purification
pTYB3, 4, pKYB1 Sce VMA C-terminus Thiol
a
[40] Purification, generation of C-terminal thioesters
a
Other nucleophiles might be used for the induction of the protein cleavage.
668 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004

segments in high yields is possible by using the introduced
IMPACT
TM
-system. Thioesters can be obtained by fusing
the protein of interest with the N-terminus of an intein,
proteins with N-terminal Cys by fusing with the C-terminus
of a mutated intein [64]. Both fragments needed for ligation
can be synthesized alternately by SPPS as described already,
so it is possible to introduce specific labels either at the
N- or C-terminus of the protein. The chemically synthesized
section can be as small as possible whereas the expressed
part is not limited in size. This can lead to very large
specifically labelled proteins.
Expressed protein ligation can be performed directly on
chitin beads and thiolysis and ligation can occur simulta-
neously. It is disadvantageous if solubilizing agents are
needed for the ligation, because urea or guanidinium
hydrochloride for example denaturate the chitin binding
domain at concentrations higher than 2
M
. Alternatively,
the thioester may be eluted and the ligation reaction may
proceed in a second step. Detergents, urea or guanidinium
hydrochloride can be used in higher concentrations to
increase the solubility of peptides which may result in a
higher reaction yield.
If an amino acid within the protein sequence or several
amino acids on both ends was to be modified, the
protein would have to be split in three or more fragments
andtwoormoreligationstepswouldhavetobe

Semisynthesis of prohormones proNPY [37,75,110]
Prenylation of proteins Rab7, YPT1 GTPase [114,115]
In vitro cyclization c-Crk-II [116]
Protein cyclization in vivo GFP [92]
Semisynthesis of cytotoxic proteins RNase A [63]
Incorporation of non natural amino acids Src [67]
Peptide and protein labelling with biophysical probes c-Crk-II, hIL-8 [73,76]
Conditional splicing in vivo MBP [83,84]
Cyclization using the TWIN system BBP, RGD [88]
In vitro screening for splicing inhibitors GFP [117]
Ó FEBS 2004 Expressed protein ligation (Eur. J. Biochem. 271) 669
The Csk–Src system was also investigated by Wang et al.
who displaced the Src–tyrosine by five unnatural Tyr
analogues to determine the role of the Tyr-sidechain for
Src affinity to Csk [67]. Lu et al. [68] observed the
influence of phosphorylation at two Tyr residues of
protein tyrosine phosphatase SHP-2 by introducing non-
hydrolyzable phospho-tyrosine analogues at the phos-
phorylation site of SHP-2 by expressed protein ligation.
Their results showed that phosphorylation at Tyr542 leads
to the basal inhibition of protein tyrosine phosphatase
(PTPase) activity by interacting with the N-terminal SH2
domain, whereas phosphorylated Tyr580 stimulates the
PTPase by interacting with the C-terminal SH2-domain.
The role of phosphorylation of the eukaryotic initiation
factor elF4E, which is implicated in the regulation of the
initiation step of translation, was observed by the
selectively phosphorylated version. Cap affinity of phos-
phorylated and unphosphorylated elF4E was determined
by fluorimetric time-synchronized titration [69].

bridges was necessary to obtain biological activity of hIL-8.
One of these Cys residues was chosen as a ligation site.
Internalization studies on HL60-cells expressing both
hIL-8-receptor subtypes and binding studies on HL60-
membranes provided an insight into the ligand receptor
interaction and the internalization of the interleukin-8-
receptor complex [76].
Also, single atoms like isotopes or atom homologues like
F instead of H, or Se instead of S can represent biophysical
probes. Wallace et al. introduced simultaneously (and site-
specific) selenium and bromine as reporter atoms into the
sequence of cytochrome c without significant changes of
structure and function [77].
Intermolecular protein splicing in
trans
to study
protein–protein interaction
Protein–protein interactions are essential for many biologi-
cal processes like receptor-ligand binding, protein polymer-
ization, gene expression, etc. To study these interactions
in vivo, several methods have been developed, one example
being the yeast two-hybrid system. The principle of these
methods is that potentially interacting proteins are tagged to
proteins with a particular function [78]. This function will be
recovered if an interaction of the tagged proteins is
accomplished. By using protein-splicing in trans [79] a split
intein is tagged to a split functional protein that is
reconstituted after interaction of the intein parts. Ozawa
et al. used halves of enhanced green fluorescent protein
(eGFP) as N- and C-terminal exteins and fused them to

shuttle-ability of hCT and its possible role in drug delivery
was demonstrated using eGFP [86].
Generation of cyclic peptides and proteins
Backbone cyclization can improve the stability and the
activity of peptides and proteins and reduce their conform-
ational flexibility. The production of circular proteins may
influence the rational design of enzymes and the develop-
ment of new agents by structure activity studies.
Cyclic structures can be obtained either by disulfide
formation or by formation of a peptide bond between
N- and C-termini or by sidechain cyclization. Several
methods have been developed by using modified inteins to
generate cyclic peptides and proteins. The aim is to create a
protein with both an N-terminal Cys and a C-terminal
670 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004
thioester. Such a peptide can be generated by flanking the
protein of interest with two inteins (Fig. 7). The N-terminal
modified intein can be cleaved by a pH and temperature
shift, whereas the C-terminal intein is cleaved by the
addition of thiols. This ÔtwointeinsystemÕ (TWIN) also
allows the separation by chitin binding domains fused to the
inteins. The reaction of the two reactive groups leads to the
formation of cyclic peptides and proteins or multimers by
an amide bond [87,88].
Several approaches use intramolecular trans-splicing for
the generation of cyclic backbones in vivo and in vitro. In
these cases, the split intein is not coupled to a cleaved
protein or to two proteins which should be knotted, but the
intein parts flank one protein with an N-terminal Cys
residue. If the intein is reconstituted, a thioester intermediate

joined with a fragment synthesized by SPPS that contained
a naturally occurring Cys residue at the N-terminus.
Ligation of both enzymatic inactive protein segments led
to the full length protein, which reconstituted its enzymatic
activity after several renaturation steps. Another intein-
based approach was used to purify the cytotoxic endonuc-
lease I-TevI by insertional inactivation followed by pH
controllable splicing [94]. In this case, a mini intein mutant
(DI-SIM) of the full length Mtu RecA intein was inserted
into the I-TevI sequence thereby inactivating the protein
in vivo. The intein triggered the splicing of the protein after
purification on a chitin column and the endonuclease could
be obtained in its native state. However, this method was
only successful when an appropriate Cys residue was in the
target protein allowing proper insertion of the intein.
Furthermore, the toxicity has to be low and the splicing
ratio in vitro/in vivo has to be as high as possible. Expression
of the whole protein is one of the big advantages in this
system as the folding of the endonuclease does not interfere
with the folding of the intein module. Intein-based trans-
splicing systems with either native or artificial split inteins
also seem to be adequate workhorses for the synthesis of
cytotoxic proteins [91,95].
Segmental isotopic labelling
Expressed protein ligation is of great use for the introduc-
tion of stable isotopes into protein segments (Fig. 8) [96,97].
This approach circumvents the practical size limitation
for structure determination by using NMR spectroscopy.
Generally, inadequate loss of structure resolution is based
on several effects that are proportional to the number of

part of the protein of interest was bacterially expressed in
15
N-isotope containing media. Fusion of this labelled
segment with the other recombinant protein part that was
unlabelled led to the specifically labelled protein. One of the
great advantages of these labelling strategies is the possibi-
lity to elucidate particular interactions of protein domains.
Such a phenomenon could be shown in bacterial sigma
factor [101]. In this case, the comparative NMR studies of
isotopic labelled model proteins of this protein obtained by
applying EPL revealed that the C-terminal DNA binding
domain does not interact directly with the N-terminal
autoregulatory domain. EPL and trans-splicing also have a
great impact in the preparation of labelled internal protein
segments. Yamazaki’s group presented a method for central
segmental isotopic labelling by using a tandem trans-splicing
approach [102,103]. To label an inner segment of the maltose
binding protein, the target protein was expressed as three
split intein fusion proteins. The central segment was thereby
expressed in isotope containing media as a fusion protein
with attached PI-PfuI and PI-PfuII inteins at its termini.
Consequently, the N-terminal parts of the desired protein
were expressed as fusion proteins carrying the other halves of
the split inteins. Simultaneous splicing yielded the target
protein including an inner isotopically labelled fragment.
Alternative ligation methods
The only disadvantage of NCL and EPL is the necessity of a
Cys residue or a homologue at the ligation site. The
occurrence of this amino acid in globular proteins is very
low and the insertion of additional Cys residues can alter the

where a phosphine is used to reduce an azide to an amine.
An intermediate iminophosphoran possesses a nucleophilic
nitrogen which can react with an acyl donor to form an
amide. A peptide bearing a C-terminal phosphinothioester
is coupled to another peptide with an N-terminal a-azido
group to form a peptide bond. The final product has no
residual atoms [106,107]. This ligation method may also be
combined with NCL for tandem ligation applications. The
method however, has up to now only been used for small
peptides.
Expressed enzymatic ligation
This method combines the advantages of expressed protein
ligation with the substrate mimetic strategy of protease
mediated ligation. The reverse hydrolysis potential of a
protease, e.g. Glu/Asp-specific serine protease from
Staphylococcus aureus, is used to catalyze the peptide bond
formation [108]. The limiting enzyme substrate specificity
and possible proteolysis of peptides and ligated products
is eliminated by substrate mimetics carrying a site-specific
ester leaving group at the C-terminus of the former
Fig. 8. Segmental isotopic labelling. Protein
segments are expressed in unlabelled or iso-
topically enriched media as fusion proteins
with parts of split inteins. Reconstitution of
the inteins results in trans-splicing that leads
to terminally (A) or centrally (B) labelled
proteins.
672 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004
unspecific peptide [109]. The IMPACT
TM

Fig. 9. Alternate ligation methods. NCL with Cys-mimetics (A) results in Gly at the ligation site. NCL combined with desulfurization (B) leads to an
Ala residue. Staudinger ligation (C) is applicable to each amino acid at the ligation site. EEL uses the substrate mimetic approach and an inverse
working protease. The protein thioester used for ligation can be obtained by the IMPACT
TM
method (D).
Ó FEBS 2004 Expressed protein ligation (Eur. J. Biochem. 271) 673
Acknowledgements
Some of the work discussed in this article was supported by the DFG
grant 1264-5-1/2. Furthermore we kindly acknowledge the financial
support of the DFG for projects dealing with protein ligation (GK 378
and SFB 610).
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