Báo cáo khóa học: Expressed protein ligation Method and applications docx

Beck-Sickinger Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany The introduction of noncanonical amino acids and bio-physical pr

R E V I E W A R T I C L E

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 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; IMPACTTM-system; intein; native chemical ligation

Introduction

Proteins and peptides that have been modified by

intro-ducing noncanonical amino acids, fluorescence tags, spin

resonance labels or cross-linking agents have great potential

for investigations into protein–protein interactions and can

help to elucidate protein structures.Furthermore, artificial

peptides and proteins with new properties and with a broad

range of applications can be obtained.Further interest lies

in fragmental or complete isotopic labelling for NMR

studies to determine protein structures

Solid-phase peptide synthesis (SPPS) provides the

pos-sibility of introducing noncanonical amino acids into

peptides but is restricted to peptides of up to 60 amino

acids in length.By using expression systems in bacteria or

yeast, the recombinant generation of peptides and proteins and their complete isotopic labelling has become possible [1–3].The size of the constructs is not restricted but the insertion of noncanonical amino acids is difficult [4,5].The limitation of peptide size in SPPS was circumvented by several approaches developed for the synthesis of proteins

by segment condensation [6].Liu et al.used a glycolalde-hyde peptide ester for the reaction of an unmasked aldeglycolalde-hyde with an amino-group of an N-terminal Cys or Ser to form

a thiazolidine- or oxazolidine-ring.Rearrangement of the O-acyl-ester resulted in an amide bond with a pseudoproline residue [7].In the thiol capture approach, where only Cys sidechains have to be protected, a 4-mercapto-dibenzofuran ester forms an asymmetric disulfide bond with an 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 the ester and an OfiN-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: beck-sickinger@uni-leipzig.de

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 , intein-mediated purification with an affinity chitin

binding tag; IPL, intein-mediated protein ligation; NCL, native

chemical ligation; PTPase, protein tyrosine phosphatase; SPPS,

solid-phase peptide synthesis; TROSY, transverse relaxation

optimized spectroscopy; TWIN, two intein system.

(Received 12 November 2003, revised 19 December 2003,

accepted 5 January 2004)

length; larger proteins cannot be obtained easily in one

ligation step.Multistep NCL of different peptide-segments,

however, can lead to larger proteins [11].An extension of

this NCL strategy is the expressed protein ligation (EPL)

method [12] using recombinant thioesters and/or

aCys-peptides.This review gives an overview of this method and

its applications in the past few years

Native chemical ligation

The method of native chemical ligation was introduced by

Dawson et al.[13,14] and is based on the reaction between a

thioester and the sidechain of a Cys residue – reported for

the first time by Wieland et al.[15].Two fully unprotected

synthetic peptides react to form an amide bond, so they are

connected as in the native peptide backbone.The reaction

proceeds in aqueous conditions at neutral pH.The first step

of this process is the chemoselective transthioesterification

of an unprotected peptide Ca-thioester with an N-terminal

Cys of a second peptide.The so-formed thioester

sponta-neously undergoes an SfiN-acyl transfer to form a native

peptide bond and the resulting peptide product is obtained

in the final disposition.Internal Cys residues within both

peptide segments are permitted because the initial

trans-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] All 20 amino acids 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 in excess.A simple washing step completely removes the solubilized peptides and the assembled full length protein can be cleaved from the resin

In the tandem peptide ligation approach, the NCL is applied to the synthesis of peptides and proteins requiring two or more ligation steps.NCL is combined with a pseudoproline ligation by imine capture [23], the third step can be pseudoglycine ligation [24]

In addition to Cys, related amino acids, including selenoCys [25] and selenohomoCys [26], have been reported

to work in a similar manner

Thioester formation The bottleneck in NCL is the generation of the thioester Several applications have been developed using solid-phase peptide synthesis.Most of the strategies to obtain peptide thioesters have used the Boc-strategy [13,17] because of the base-lability of the thioester.However, different attempts

in the synthesis of thioesters were performed by using the 9-fluorenylmethoxycarbonyl (Fmoc) method.In general, the Fmoc-strategy has several advantages over the Boc-strategy, the first being the milder conditions used for cleavage from the resin.To circumvent the susceptibility of the thioester linkages to nucleophiles like piperidine, used for the removal of the Fmoc-protecting group, several cocktails for deprotection have been developed, e.g., 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) with 1-hydroxy-benzotriazole (HOBt) [27], 1-methylpyrrolidine with hexamethyleneimine and HOBt [28] or DBU and HOBt [29].The final cleavage from the resin then results in the peptide thioester

Further methods were introduced that used different resins.One is based on modifications of Kenner’s sulfon-amide Ôsafety catchÕ linker [30].The C-terminus of the growing peptide chain is attached to the resin with an acid-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)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.

methylenchloride [34].Unfortunately, the alkylaluminium

thiolate method can lead to epimerization at the C-terminus

and reactions at the sidechains, e.g., sidechain thioesters and

aspartimide formation.This can be avoided by using a

weaker Lewis acid, e.g Al(CH3)3[35].A further possibility

is the synthesis of peptides on Cl-trityl-resin and the

cleavage of the fully protected peptide chain with acetic

acid and trifluoroethanol.The thioester can be obtained by

the treatment of the protected peptide with activating

reagents and thiols [36,37].After deprotection of functional

sidechains with trifluoroacetic acid, the thioester can be

easily purified by HPLC (Fig.2)

An alternative approach for the thioester synthesis of

larger peptides and proteins in high yields and purity uses

a bacterial expression system based on the intein mediated

self-splicing mechanism of precursor proteins as discussed

below

Recombinant generation of proteins

with C-terminal thioester or N-terminal Cys

Inteins and their use in protein chemistry

Inteins are internal segments of precursor proteins that

catalyze their ipso excision, in an intramolecular process

called protein splicing, with the concurrent ligation of the

two flanking external regions (N- and C-exteins) through

a native peptide bond.This finally yields the host protein

Thus, inteins are analogues of self-splicing RNA introns

The first intein was discovered in 1987 and up to now over

100 inteins are listed [38–40].The origin of inteins is not yet

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 (thio)esters that are formed by an NfiS- or an NfiO-shift are intermediates of the internal rearrangement steps of the splicing reaction.This was proven by various investigations

Fig 2 Formation of synthetic peptide

a-thio-esters Peptide a-thioesters can be synthesized

by the Fmoc strategy by using backbone

amide linker resins (A), acidic cleavage from

mercaptoalkyl linker resins (B), Lewis acid

activated cleavage from common resins

(C), cleavage of fully protected peptides

(Boc, t-butyloxycarbonyl; tBu, t-Butyl) and

deprotection after thioester generation (D)

and by using of sulfonamide safety catch

linker resins (E).

Fig 3 Characteristic positions of intein motifs and numbering The inserted intein carries the N-terminal extein (left shaded box) and the C-terminal extein (right shaded box).The residues important for the splicing process as well as the conserved segment blocks (A, B, C, D, E,

H, F, G) and some internal intein key amino acids are depicted in the one letter code within the certain segments (bold black).Numbering of the amino acids of a precursor protein is made in the following way: the intein’s N-terminal amino acid (Cys, etc.) is numbered as 1 whereas the C-terminal amino acid of the N-terminal extein is num-bered as )1 and the N-terminal residue of the C-terminal extein is numbered beginning with +1.

Replacement of the amino acid residues at the N-terminus

containing a nucleophilic thiol or hydroxyl sidechain and

the Asp at the C-terminus, through site directed

mutagen-esis, ended up in a complete loss of splicing activity of the

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 peptide bond into a twisted conformation.This could also

be protonated through the penultimate His residue men-tioned above.Mutation of this amino acid did not affect the first steps of the splicing up to the branched intermediate but abolished the final step.In the X-ray crystal structure of the intein, Mycobacterium xenopi gyrase (Mxe GyrA) (Fig.5), the His197 is hydrogen bonded to Asn198 so that His197 is oriented for the donation of a proton from Nd position to the emerging alpha amino group of the C-extein, prior

to the SfiN-acyl shift [51,52].Some putative inteins that lack the penultimate His residue are either inactive or use other amino acids.Accordingly, the penultimate His is not absolutely required but increases the splicing rate.Block B contains Thr and His that are separated through two amino

Fig 4 Mechanism of intein-mediated protein splicing In the initial step a thioester intermediate is formed by an NfiS-acyl shift

at the N-terminal Cys of the intein (Cys 1 ) Transthioesterification by a nucleophilic attack of the sidechain of the N-terminal Cys

of the C-extein (Cys +1 ) on the thioester is formed in the first step and results in a branched intermediate.Peptide bond cleavage coupled to succinimide formation of the C-terminal intein–Asp releases the intein.The knotted exteins undergo a spontaneous SfiN-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.

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

MxeGyrA [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

Both effects, the spatial constraints and the electronic

influence, lead to a reactive and accessible electrophilic

carbon of the scissile peptide bond as an acid/base catalysis

mechanism is suggested

Furthermore, divalent transition metal cations influence

the protein splicing process.It was shown for the split

inteins Ssp DnaE and the Mtu RecA that micromolar

concentrations of Zn2+ions decreased the splicing rate and

Zn2+ion concentrations in the millimolar range stopped

completely the process through chelation of key amino

acids.A similar effect was obtained for Cd2+ions [58,59]

Classification of inteins

The elucidation of the splicing mechanism and the

identi-fication of the key amino acid residues involved in the

scission and ligation of the peptide bonds facilitated the

molecular engineering of artificial inteins as tools for

different applications in protein chemistry.Currently there

are five general methods of intein usage in this field so far:

(a) modified inteins with an inducible autocatalytic cleavage

activity are used for protein purification; (b) inteins are used

for trans-splicing.Here the inteins are split into two

fragments that can recombine and reconstitute their splicing

activity in vivo or in vitro.(c) Intein mediated protein ligation

(IPL) is used for the generation of specifically

mono-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 However, the lack of the succinimide formation did not affect the preceding NfiO-acyl rearrangement at the N-terminal splicing junction.The same data were found previously for the NfiS-acyl shift in the Sce VMA intein Incubation of this modified intein with thiols, like dithio-threitol, releases the corresponding free C-terminal thioester-tagged extein from the N-terminal splicing junction through transthioesterification.This thiol-inducible cleavage activity

of an engineered intein was the beginning of the extensive exploitation of other intein mutants as workhorses in the area of biotechnology to obtain mono-thioester labelled proteins and aCys-proteins [46,50]

Fig 5 Comparison of Mxe GyrA (A) and Sce

VMA (B) intein structure The structures of

both inteins have been determined by X-ray

crystallography [51,55,56] (PDP files 1AM2

and 1LWS, http://www.rcsb.org/pdb/) Blue

arrows indicate b-sheets whereas purple

cyl-inders symbolize a-helices.The N-termini are

coloured in green and C-terminal b-sheets in

red.The endonuclease domain of Sce VMA

(right part) is clearly separated from the

self-splicing domain (left part).

The IMPACTTM-system [62] [intein-mediated purification

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 4C 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 commonly used with a AsnfiAla 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

Choice of thiols For the thiolysis of the intein fusion proteins, a broad range

of thiols have been investigated.The choice of a certain thiol depends on the accessibility of the catalytic pocket of the intein/extein splicing domain and the properties of the target protein of interest.In general, the thiols should be small, nucleophilic molecules that can enter the catalytic pocket to attack the thioester bond connecting the extein and the intein.For further application of protein thioesters in EPL two things have to be considered to be dependent on the synthesis strategy.On one hand, the protein thioester should

be stable to hydrolysis in order to be isolated.On the other hand, the thioester should also be reactive enough in EPL Simple alkyl thioesters are quite stable to hydrolysis but not very reactive.Mixtures of alkylthiols and thiophenol [12,19]

or 2-mercaptoethansulfonic acid (MESNA) [63] improved the reactivity.If there is no need for a thioester isolation, MESNA or thiophenol could be used directly for the induction of the cleavage and the subsequent reaction Instead of thiols, other nucleophiles like hydroxylamine [45] can also be used to induce protein splicing and the isolation

of the target protein

Fig 6 Expressed protein ligation The synthesis of proteins with

C-terminal thioester (left) and proteins with N-terminal Cys (right) can

be performed by using the IMPACTTM-system.Thioesters can be

obtained by fusing the protein of interest to the N-terminus of an

intein, proteins with N-terminal Cys by fusing to the C-terminus of a

mutated intein.Separation occurs by using the Chitin binding domain

(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 Thiola [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 Thiola [40] Purification, generation of C-terminal thioesters

a Other nucleophiles might be used for the induction of the protein cleavage.

Generation of aCys proteins

The EPL requires a peptide or protein that contains an

amino terminal Cys residue (aCys) besides the a-thioester

moiety.To synthesize proteins possessing an aCys, the

protein cDNAs of interest can be cloned into various

commercially available vectors as mentioned above

(IMPACTTM-System).Thus, after the expression, the

intein/CBD fusion protein can be purified on a chitin

column and cleaved by changing the pH or temperature

This will lead to the free aCys proteins.One drawback in

the intein-based synthesis of aCys proteins is the possible

spontaneous cleavage, which results in a loss of the

purification tag [45,64]

Expressed protein ligation (EPL)

Expressed protein ligation [12,50,65,66], also named

intein-mediated protein ligation [46], is an extension of the NCL

method.A recombinant Ca-thioester reacts with a

chemi-cally synthesized or expressed peptide/protein possessing an

N-terminal Cys under the conditions of NCL to form a

native peptide bond.This ligation method combines the

advantages of molecular engineering and chemical peptide

synthesis in many cases and allows site-specific introduction

of unnatural amino acids and chemical or biophysical tags

into large proteins.In former times, the difficulty of this

strategy was the chemical preparation of peptides or

proteins with a C-terminal thioester and the generation of

peptides and proteins with N-terminal Cys residues in large

quantities and high purity.Now, the expression of both

segments in high yields is possible by using the introduced

IMPACTTM-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 2M.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 and two or more ligation steps would have to be executed.The second peptide fragment carrying an N-terminal Cys and an additional C-terminal thioester has to be masked recombinantly at the N-terminus with

a protease cleavage site, e.g factor Xa protease After the first ligation step, the N-terminal Cys is liberated by protease treatment and the second ligation step can be performed [50].This protein can be synthesized from the C- to N-direction

Applications of expressed protein ligation

EPL chemistry applications are summarised in Table 2 and described in more detail below

Site specific protein modifications The ability to change specific sidechains by the insertion of noncanonical amino acids has great potential in protein structure/function studies

To determine the role of post-translational modifica-tions it is necessary to insert phosphorylamodifica-tions or glyco-sylations at defined positions.A phosphotyrosine peptide

is ligated to the C-terminus of the protein tyrosine kinase C-terminal, Src kinase (Csk), which results in an intra-molecular phosphotyrosine–Src homology 2 interaction and increased catalytic phosphoryl transfer to a substrate when compared with a nonphosphorylated control [12]

Table 2 Recent highlights show the scope of EPL chemistry GFP, green fluorescent protein; CAR D1, immunoglobulin D1 domain of cox-sackievirus-adenovirus receptor; MBP, maltose binding protein; proNPY, proneuropeptide Y; BBP, brain-binding peptide; RGD, (Arg-Gly-Asp)-containing peptide.

Investigation of protein–protein interactions Enhanced GFP [78,80,81]

Peptide and protein labelling with biophysical probes c-Crk-II, hIL-8 [73,76]

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 unphosphos-phorylated elF4E was determined

by fluorimetric time-synchronized titration [69]

The introduction of biophysical probes (spin labels or

fluorescence tags) allows the observation of protein–protein

interactions, membrane insertion or cellular uptake of

labelled peptides and proteins.Several fluorescence based

approaches [70–72] have been developed where the

fluoro-phore is attached to the sidechain of an amino acid (mainly

Lys) within the protein sequence

Cotton et al.described the synthesis of a dual-labelled

version of the Crk-II adapter protein and its investigation

by fluorescence resonance energy transfer (FRET).A pair

of tetramethylrhodamine and fluoresceine was ligated to

the N- and C-terminus by solid-phase expressed protein

ligation.The construct reported the phosphorylation of

Crk-II by the nonreceptor tyrosine kinase by fluorescence

change that was affected by structural changes [73].The

same FRET-pair was used to observe

homo-oligomeriza-tion of glutathione S-transferase, SH2 domain

phospha-tase-1 and serotonin N-acetyltransferase by measurement

of intermolecular FRET-effects [74].We succeeded

recently in the semisynthesis of the 69 amino-acid

proNPY and its analogues to study prohormone

proces-sing.Five variants were synthesized containing either no

label or were labelled with carboxyfluorescein or biotin

Western blot analysis was performed to determine the

binding site of anti-NPY and anti-proNPY antibodies

[75]

Furthermore we synthesized human interleukin-8, a

chemotactic cytokine, and its C-terminal

carboxyfluo-rescein-labelled analogue by expressed protein ligation

Possessing four Cys residues, the formation of two

disulfide-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 intrans 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 N- and C-terminal fragments of a modified intein [80,81]

No fluorescence was observed from any construct expressed

in E.coli.In contrast, coexpression of calmodulin and its target peptide M13 connected to the intein led to fluores-cence of eGFP, suggesting that the interaction of calmo-dulin and M13 triggers the refolding of the intein.A related approach using firefly luciferase, was introduced by the same group for mammalian cells [82]

The conditional protein splicing approach from Mootz

et al.[83,84] used the dimerization of the rapamycin receptor FKBP and the rapamycin binding domain in the presence of rapamycin to reconstitute a split intein in mammalian cells.Maltose binding protein (MBP) and a His-tag were used as exteins and the splicing product was detected by Western blotting or by immunoprecipitation in the cells.In a related approach by this group, GFP was coupled to the N-terminus of an intein and expressed in Chinese hamster ovary cells.The chemically synthesized C-terminal part of the intein was coupled to a FLAG-epitope and transported through the membrane by using a protein transduction domain.The C-terminal intein can associate with its N-terminal half within the cells and ligation of GFP to the FLAG-epitope is performed [85]

By using the EPL-method, eGFP was ligated to an amidated human calcitonin (hCT) derived carrier peptide Covalently bound calcitonin and its C-terminal fragments were shown to permeate membranes of nasal epithelium, but permeation was limited to peptides.Ligated eGFP-hCT(8–32) shows specific mucosal internalization, whereas enhanced GFP did not show internalization per se.The 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

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 Ôtwo intein systemÕ (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

will be formed that undergoes

transthioesterification.Cyc-lization of Asp and SfiN-acyl transfer leads to a cyclic

product [89–92]

A simple approach for in vivo cyclization in Escherichia

colicells was introduced by Camarero et al.[93].An SH3

domain from murine c-Crk adapter protein with an

N-terminal Cys residue was N-terminally fused to an intein

with a chitin binding domain.After the expression of this

fusion protein, the N-terminal Met residue produced by the

start-codon is replaced by the Met-aminopeptidase, which

results in an active Cys residue.The amide-bond connecting the protein to the intein can switch by NfiS-acyl shift to the thioester bond.As this protein now possesses a reactive N-terminal Cys residue and a C-terminal thioester it can react to form an intramolecular bond by NCL

Generation of cytotoxic proteins

In some cases, the expression of the desired proteins in bacteria can cause cytotoxic side-effects because the target protein competes with cellular components of the host Another problem is that overexpressed proteins may aggregate as inclusion bodies in the cytosol.By using EPL techniques this can be avoided through modular synthesis of

an artificial target protein as an intein fusion protein Subsequently, through ligation and refolding, the native conformation and biological functionality of a cytotoxic protein will be recovered.The potential cytotoxic RNase A was expressed by this method [63].One part of this protein was produced as a segment carrying an intein at its C-terminal site.After thiol-induced intein-mediated clea-vage, the obtained thioester of the truncated RNase A was 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 amino acids.This includes line broadening, longer rota-tional correlation times and an increased number of signals

of similar chemical shifts.Even though there are new NMR techniques available, like transverse relaxation optimized spectroscopy (TROSY) [98], the standard isotopic labelling strategies through incorporation of uniformly labelled15N,

13C and perdeuteration of amino acid sidechains bear the

Fig 7 Generation of cyclic proteins Intramolecular trans-splicing

(left).The two parts of a split intein flank one protein with N-terminal

Cys.If the intein is reconstituted, a thioester intermediate will be

produced that undergoes transthioesterification.After Asp cyclization

and SfiN-acyl transfer, a cyclic product is formed.Two intein (TWIN)

system (right).The protein of interest is cloned between two inteins.

The N-terminally modified intein can then be cleaved with a pH and

temperature shift, whereas the C-terminal intein is cleaved by addition

of thiols.The reaction of the two reactive groups leads to the formation

of cyclic peptides and proteins.

signal overlap of macromolecular systems.Yamazaki et al.

selectively labelled the C-terminal domain of the E coli

RNA polymerase a-subunit [99] by using a trans-splicing

system based on a split PI-PfuI intein.Muir and coworkers

used the EPL strategy to introduce an15N-labelled domain

within the Src-homology domain 3 and 2 segment derived

from AbI protein tyrosine kinase [100].In both cases the

part of the protein of interest was bacterially expressed in

15N-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

protein structure and function by the formation of disulfide

bridges.Several approaches have been developed to

circumvent this limitation (Fig.9)

NCL with Cys-mimetics

The NCL-methodology has been extended to -X-Gly- and

-Gly-X- ligation sites [104].One peptide possessing a

C-terminal thioester reacts with a second one containing

either an Na(ethanethiol) peptide or a Na(oxyethanethiol) peptide.The thioester intermediate forms a 5- or 6-member ring and in a final SfiN-transfer an amide bond is formed

In a subsequent step, the substitution at the amide bond can

be removed by the treatment with Zn and H+to form a native peptide bond

NCL combined with desulfurization

In this application, NCL is extended to proteins without Cys-residues [105].Ala is a common amino acid in peptides and proteins, thus, a specific Ala is replaced by a Cys residue

at the ligation site within the sequence of the protein of interest.Then NCL is performed to ligate thioester and Cys-peptide.In the following step the Cys is converted to an Ala

by desulfination using palladium or Raney-nickel and hydrogen.This approach can be used for the synthesis of linear and cyclic proteins and extends NCL-methodology

to -X-Ala-.As no selectivity of the desulfurization reaction

is possible, proteins that contain further Cys residues cannot

be made by this technique

Staudinger ligation This ligation method is inspired by the Staudinger reaction, 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.