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

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 -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 aldehyde 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 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: 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) Eur. J. Biochem. 271, 663–677 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03978.x 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].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 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 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 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(CH 3 ) 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. Ó FEBS 2004 Expressed protein ligation (Eur. J. Biochem. 271) 665 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. 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. 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 Zn 2+ ions decreased the splicing rate and Zn 2+ ion concentrations in the millimolar range stopped completely the process through chelation of key amino acids. A similar effect was obtained for Cd 2+ 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). Ó FEBS 2004 Expressed protein ligation (Eur. J. Biochem. 271) 667 IMPACT TM -system The IMPACT TM -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 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. 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 IMPACT TM -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 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 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 (IMPACT TM -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 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 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 phosphorylations 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. Application Subject References Investigation of protein–protein interactions Enhanced GFP [78,80,81] Targeting CAR D1, calcitonin segment [86,113] Internal isotopic labelling MBP [102] 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]. 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 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 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 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 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 coli cells 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 labelled 15 N, 13 C 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. Ó FEBS 2004 Expressed protein ligation (Eur. J. Biochem. 271) 671 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 an 15 N-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 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 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 beremovedbythetreatmentwithZnandH + 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. 672 R. David et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... FEBS 2004 Expressed protein ligation (Eur J Biochem 271) 673 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. .. 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Keywords: expressed protein ligation; IMPACT TM -system; intein; native chemical ligation. Introduction Proteins and. expressed protein ligation (EPL) method [12] using recombinant thioesters and/ or aCys- peptides. This review gives an overview of this method and its applications