Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.

FEBS Letters xxx (2015) xxx–xxx

journal homepage: www.FEBSLetters.org

Review

Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis Robert B. Quast, Devid Mrusek, Christian Hoffmeister, Andrei Sonnabend, Stefan Kubick ⇑ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany

a r t i c l e

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Article history: Received 16 March 2015 Revised 17 April 2015 Accepted 21 April 2015 Available online xxxx Edited by Wilhelm Just Keywords: Non-standard amino acids Cotranslational incorporation Cell-free protein synthesis Eukaryotic systems

a b s t r a c t Over the last years protein engineering using non-standard amino acids has gained increasing attention. As a result, improved methods are now available, enabling the efficient and directed cotranslational incorporation of various non-standard amino acids to equip proteins with desired characteristics. In this context, the utilization of cell-free protein synthesis is particularly useful due to the direct accessibility of the translational machinery and synthesized proteins without having to maintain a vital cellular host. We review prominent methods for the incorporation of non-standard amino acids into proteins using cell-free protein synthesis. Furthermore, a list of non-standard amino acids that have been successfully incorporated into proteins in cell-free systems together with selected applications is provided. Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction Nature has evolved a set of 20 primary building blocks – the proteinogenic or standard amino acids (sAAs) – serving as substrates in ribosome-mediated protein synthesis in almost all living organisms. Exceptionally, this natural repertoire is expanded by selenocysteine [1] and pyrrolysine [2,3] that are translationally incorporated only in certain species. Based on their biochemical and biophysical characteristics this finite number of amino acids limits the ability to directly study proteins but thereby defines the possible chemical space of protein chemistry [4]. Chemical synthesis bears the potential to generate a vast variety of non-standard amino acids (nsAAs) with a tremendous amount of possible characteristics exceeding the natural repertoire of primary protein building blocks [5,6]. With the objective of supporting biological investigations as well as creating proteins with novel characteristics scientists have developed methods in order to cotranslationally incorporate a variety of different nsAAs into Abbreviations: aatRNA, aminoacyl-tRNA; CECF, continuous-exchange cell-free; CFCF, continuous-flow cell-free; CFPS, cell-free protein synthesis; CuAAC, copper-catalyzed alkyne-azide cycloaddition; eRF1, eukaryotic release factor 1; IRES, internal ribosome entry site; nsAA, non-standard amino acid; oRS, orthogonal aminoacyl-tRNA synthetase; otRNA, orthogonal tRNA; OTS, orthogonal translation system; Pyl, pyrrolysine; PylRS, pyrrolysyl-tRNA synthetase; RF, release factor; sAA, standard amino acid ⇑ Corresponding author. Fax: +49 331 58 187 119. E-mail address: [email protected] (S. Kubick).

proteins. In the following we review some of the most prominent approaches with special emphasis on their use in the context of cell-free protein synthesis (CFPS). Although the methods presented here have mainly been applied in Escherichia coli based cell-free systems (for a more concise presentation please refer to [7] and [8]) this article aims at pointing out key features that support their transfer to eukaryotic CFPS. Finally we provide an overview on diverse nsAAs that have been successfully employed in different CFPS systems (Fig. 1 and Table 1) together with a presentation of selected applications. 2. Cell-free protein synthesis CFPS also termed in vitro protein translation has emerged as a versatile technology to complement cell-based protein expression [9–12]. CFPS utilizes the translational machinery of cells obtained either by preparation of crude cell extracts or the reconstitution of purified components. By addition of an appropriate template that can be either DNA or mRNA protein synthesis is directed towards the predominant production of a desired protein. Until today CFPS based on a variety of different sources including E. coli [13–16], Leishmania [17], Thermus [18], wheat germ [19,20], tobacco [21], insect [22–24], yeast [25], rabbit reticulocytes [26], CHO [27], mouse [28] and human cells [29,30] has been demonstrated. Different reaction modes are available that enable transcription and translation to be performed separately (linked

http://dx.doi.org/10.1016/j.febslet.2015.04.041 0014-5793/Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Quast, R.B., et al. Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.04.041

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R.B. Quast et al. / FEBS Letters xxx (2015) xxx–xxx Table 1 List of nsAAs that have been incorporated using cell-free protein synthesis. The entry numbers correspond to numbers given in Fig. 1. It should be noted that this list is not intended to be exhaustive. Abbreviations used: rs = residue-specific; pc = precharged tRNA; op = orthogonal tRNA/synthetase pair; fs-tRNA = frame-shift-tRNA; s-tRNA = stop-codon-suppressor-tRNA; RR = rabbit reticulocyte; WG = wheat germ. Entry 1 2 3 4 5 6 7 8

Non-standard amino acid

Methodology

4-Fluorotryptophan 4-Methyltryptophan 5-Fluorotryptophan 5-Hydroxytryptophan 5-Methyltryptophan 6-Fluorotryptophan 6-Methyltryptophan 7-Azatryptophan

rs (E. coli [80]) rs (E. coli [80]) rs (E. coli [80]) pc s-tRNA (E. coli [181]) rs (E. coli [80]) rs (E. coli [80]) rs (E. coli [80]) rs (E. coli [80]); pc fs-tRNA (E. coli [162]); pc s-tRNA (E. coli [126,181]) rs (E. coli [130,178]) pc fs-tRNA (E. coli [111]; Sf21 [115]) pc fs-tRNA (E. coli [132]) op s-tRNA (E. coli [182]) pc s-tRNA (E. coli [126,181]) pc s-tRNA (E. coli[125]) pc s-tRNA (E. coli [125]); pc fs-tRNA (E. coli [111,113,164]; RR [113]; Sf21 [115]) pc s-tRNA (E. coli [125]) op s-tRNA (E. coli [127]) pc s-tRNA (E. coli [183]) op s-tRNA (E. coli [63,108,128,169,171,172,178,179]; Sf21 [107]) op s-tRNA (E. coli [177]) op s-tRNA (E. coli [95,128,171]) pc s-tRNA (E. coli [126,128]; RR [175]); pc fs-tRNA (E. coli/RR [113]) op s-tRNA (E. coli [128,145]) op s-tRNA (E. coli [171]) op s-tRNA (WG [154]) pc s-tRNA (WG [124]) pc s-tRNA (WG [170]); op s-tRNA (E. coli [173]) op s-tRNA (E. coli [63,95,108,174,176,178]) pc s-tRNA (E. coli [184]) rs (E. coli [130,178,179]) rs (E. coli [155]) pc s-tRNA (RR [185]); pc fs-tRNA (E. coli [186] rs (E. coli [187]) pc s-tRNA (E. coli/RR [167]) pc s-tRNA (E. coli/RR [167]) pc s-tRNA (E. coli [162]) pc fs-tRNA (E. coli [133,164]) op s-tRNA (E. coli [127,128,158]) pc fs-tRNA (E. coli [156,159,188]); s-tRNA (E. coli [188]) pc fs-tRNA (E. coli [188]) pc fs-tRNA (E. coli [156,163])

9 10 11 12 13 14 15

Azidohomoalanine 2-Naphthylalanine 2-Anthrylalanine Acetyl-lysine Dansyl-lysine Homophenylalanine para-Nitrophenylalanine

16 17 18 19

para-Fluorophenylalanine para-Trifluoromethylphenylalanine para-Iodophenylalanine para-Azidophenylalanine

20 21 22

para-Azidomethyl-phenylalanine para-Acetylphenylalanine para-Benzoylphenylalanine

23 24 25 26 27 28 29 30 31 32 33 34–39 40–45 46 47 48 49 50 51

para-Bipyridylphenylalanine 2-Methyltyrosine 3-Iodotyrosine 3-Chlorotyrosine 3-Azidotyrosine Propargyloxyphenylalanine Allylglycine Homopropargylglycine Selenomethionine Biocytin and derivatives L-3,4-Dihydroxyphenylalanine Glycosylated serine derivatives Glycosylated tyrosine derivatives Dabcyl-diaminopropionic acid b-Anthraniloyl-L-a,b-diaminopropionic acid (7-Hydroxy-coumarin-4-yl)ethylglycine Bodipy-FL-aminophenylalanine and derivatives Bodipy558-aminophenylalanine 4-[(6-[Tetramethylrhodamine-5-(and-6)carboxamido]hexanoyl)amino]phenylalanine

mode) or simultaneously (coupled mode) in one reaction vessel [8]. Even linear PCR products can be used as gene templates significantly reducing time expenses by circumventing laborious cloning steps [31–33]. Lacking the cellular plasma membrane the open environment of CFPS enables the direct manipulation of the protein translation process to achieve a tailored protein production. Therefore, CFPS is a promising tool for the synthesis of difficult-to-express proteins [8,34]. For example, the over-expression of toxic and membrane proteins is strongly facilitated due to the fact that CFPS lacks constrains accompanied by cell vitality. In general, the choice of appropriate synthesis conditions has to be determined in accordance with the individual protein to be expressed in order to obtain maximum yields of properly folded and functional products. In this context, prokaryotic systems are characterized by comparatively high protein yields but due to the limited capability to perform sophisticated post-translational modifications e.g. phosphorylation, glycosylation and signal peptide cleavage, the synthesis of functional eukaryotic proteins can be improved using cell-free systems based on extracts derived from cultured eukaryotic cells.

2.1. Unique features of eukaryotic cell-free systems Over the last years, systems derived from wheat germ embryos have become the most recognized eukaryotic CFPS systems (for a detailed overview please refer to [35]). A multitude of different proteins have been successfully synthesized using wheat germ extracts, illustrating its versatility and in particular the convenience of a eukaryotic translation and folding machinery. Nevertheless, cell-free wheat germ systems lack the capability to perform post-translational modifications, which can clearly have a significant impact on protein folding and corresponding functionality. Interesting developments in the extract preparation procedure have enabled the production of eukaryotic cell-free systems based on crude extracts from cultured tobacco BY-2 [21], Sf21 [36,37], CHO and human K562 [38] cells that contain endogenous microsomal structures. These lipid bilayers have been found to carry out some of the functions of the endoplasmic reticulum thereby providing PTMs such as protein N-glycosylation [37,39], lipid modifications [40] and signal peptide cleavage [41]. They further support active cotranslational translocation of proteins


R.B. Quast et al. / FEBS Letters xxx (2015) xxx–xxx

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Fig. 1. Non-standard amino acids that have been incorporated in cell-free protein synthesis systems. 1–8 Tryptophan-analogs: (1) 4-fluorotryptophane (4FW); (2) 4methyltryptophane (4MW); (3) 5-fluorotryptophane (5FW); (4) 5-hydroxytryptophane (5OHW); (5) 5-methyltryptophane (5MW); (6) 6-fluorotryptophane (6FW), (7) 6methyltryptophane (6MW); (8) 7-azatryptophane (7azaW). 9–11 Alanine-analogs: (9) azidohomoalanine (Aha); (10) 2-naphthylalanine (2NpA); (11) 2-anthrylalanine (AntA). 12–13 Lysine-analogs: (12), acetyl-lysine (AcK); (13) Dansyl-lysine (DaK). 14–23 Phenylalanine-analogs: (14) homophenylalanine (hF); (15) para-nitrophenylalanine (pNF); (16) para-fluorophenylalanine (pFF); (17) para-trifluoromethylphenylalanine (tFmF); (18) para-iodophenylalanine (IoF); (19) para-azidophenylalanine (AzF); (20) para-azidomethylphenylalanine (AzMeF); (21) para-acetylphenylalanine (AcF); (22) para-benzoylphenylalanine (Bpa); (23) para-bipyridylphenylalanine (Bpy). 24–28 Tyrosine-analogs: (24) 2-methyltyrosine (2MeY); (25) 3-iodotyrosine (3IY); (26) 3-chlorotyrosine (3ClY); (27) 3-azidotyrosine (3AzY); (28) propargyloxyphenylalanine (Ppa). 29–30 Glycine-analogs: (29) allylglycine (AlG); (30) homopropargylglycine (HpG). 31–33 Other non-standard amino acids: (31) selenomethionine (SeM); (32) biocytin (Bct); (33) L-3,4-dihydroxyphenylalanine (L-DOPA). 34–39 Glycosylated serine-analogs; 40–45 glycosylated tyrosine-analogs (see reference for structural details). 46–47 Quencheramino acids: (46) dabcyl-diaminopropionic (Dbc); (47) b-anthraniloyl-L-a,b-diaminopropionic acid (Adp). 48–51 Fluorescent amino acids: (48) (7-hydroxy-coumarin-4yl)ethylglycine (Hco); (49) bodipy-FL-aminophenylalanine (BODIPY-FL-Phe); (50) Bodipy558-aminophenylalanine (BODIPY558-Phe); (51) 4-[(6-[tetramethylrhodamine-5(and-6)-carboxamido]hexanoyl)amino]phenylalanine (TAMRA-X-Phe).

containing an appropriate signal peptide into the lumen of these microsomes [42]. This nature like compartmentalization was found to facilitate the correct formation of disulfide bonds yielding a set of different functional single chain antibody fragments [43]. Moreover, embedment of integral membrane proteins into these natural membranes provides a platform for the direct functional characterization in a nature-like environment [37,44]. 2.2. Improving yields and scaling eukaryotic CFPS Unfortunately, the majority of eukaryotic cell-free systems available suffer from comparatively low total protein yields. Exceptions are CFPS systems based on extracts from wheat germ

embryos. These systems provide the production of milligram amounts of target proteins within a few days [35]. Different approaches that have facilitated the development of high-yielding systems from wheat germ embryos have been found to be transferable to other eukaryotic systems. For instance, the utilization of viral internal ribosome entry sites (IRES) has just recently been demonstrated to increase translation rates in cell-free yeast [45], insect, CHO and human systems [46]. In particular, IRES sequences that provide a translation initiation independent of the 50 -cap structure such as the cricket paralysis virus intergenic region IRES thereby circumvent the major bottleneck of eukaryotic translation initiation in CFPS [47]. Moreover, different reaction formats such as continuous exchange (CECF) and continuous flow (CFCF) have


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been developed which provide a prolonged reaction time and increased protein yields [48]. In particular, the CECF approach bears the potential to be widely employed due to an easy-to-handle methodology that is based on a simple dialysis chamber composed of a reaction and a feeding compartment separated by a semi-permeable membrane. In a recent study, its compatibility with the before-mentioned microsome-containing Sf21 system was demonstrated [24]. Additionally, the CECF approach has been shown to be scalable up to several milliliters of reaction volume using a cell-free system derived from HeLa cells [49].

aatRNAs [71,72] and also steric hindrance by adjacent amino acid side chains seems to play an important role [37]. Alternatively, Suga and coworkers have evolved ribozymes catalyzing the aminoacylation of tRNAs in a versatile, rather than a specific manner [73]. Further improvements of their technology have yielded catalytic RNAs denoted as Flexizymes, offering a broad tRNA and amino acid substrate specificity [74]. Immobilized on a resin, they enable an easy-to-handle methodology to obtain aatRNAs that can subsequently be used for the incorporation of nsAAs into peptides and proteins in cell-free synthesis [75–77].

3. Methods for cell-free nsAA incorporation

3.3. Sense codon reassignment

3.1. The genetic code and its expansion

In principle, any of the 61 sense codons can be reassigned to incorporate nsAAs resulting in a residue-specific replacement of a sAA. The relative amount of residue-specific nsAA incorporation into protein can be partial to quantitative. Depending on the frequency of the codons employed, this methodology either leads to a single nsAA or multiple nsAAs in the translated protein [78]. In the 1950s, Cohen and coworkers demonstrated the tolerance of the translational machinery towards analogues of the sAAs by globally replacing Met by SeMet in E. coli [79]. In the context of cell-free protein synthesis, this requires extract preparation from auxotrophic strains [80] or depletion of the entire sAA pool by size-exclusion chromatography during extract preparation [27]. Subsequently, the reaction is supplemented with the desired repertoire of amino acids including the nsAAs but missing the sAA to be replaced. In contrast, the addition of a nsAA to the pool of all canonical amino acids induces a competition between one of the sAAs and its non-standard analogues resulting in a statistical incorporation of the nsAA depending on the relative ratio of the two types amino acid species. In the same manner pre-charged tRNAs can be used to introduce nsAAs, which is particularly useful if no aaRS accepting the desired nsAA is available. Accordingly, in the presence of the complete endogenous pool of tRNAs the nsAA is incorporated statistically as long as it is compatible with the translation machinery. This is due to a competition of aatRNAs decoding the same codons [39,44,81]. If an efficient and site-directed incorporation is desired, the use of exogenous tRNAs acting orthogonal to the host system is required. Thus, the tRNA may not be aminoacylated or deacylated by endogenous aaRSs. In the first instance, tRNA orthogonality arises from significant structural differences between the three domains of life [82,83]. Nevertheless, further efforts have been made to enhance orthogonality by tRNA evolution [84]. To achieve a global reassignment of a selected codon cell-free extracts can also be depleted of endogenous tRNAs, followed by a subsequent supplementation with only the desired tRNA species [85,86]. In theory, this would enable the use of multiple codons for the parallel incorporation of different nsAAs. Since cell-free protein synthesis is programmed by the addition of an appropriate gene template (either DNA or mRNA depending on the reaction mode), site-directed mutagenesis can be integrated during template generation to control the frequency and location of codons used for nsAA incorporation. Appropriate templates can be obtained either by gene synthesis or simply by PCR using mismatch primer pairs as the linear DNA products can directly be employed in CFPS. In particular, rare codons are considered to be promising targets for reassignment in order to expand the natural repertoire of sAAs [87]. Among the cell-free synthesis systems currently available, the PURE system represents the only system, which is completely reconstituted from recombinantly tagged protein factors purified to homogeneity. This E. coli-based system is an ideal candidate for the substitution of amino acids, tRNAs, aaRSs and other translational components that support nsAA incorporation [88]. In contrast, the complete reconstitution of a cell-free systems from

In the cellular context, proteins are synthesized based on the information defined in the genetic code. This information arises from the sequential alteration of triplet codons composed of the four bases A, G, C and T [50]. With 61 sense codons coding for the 20 sAAs and 3 non-sense codons providing a signal to terminate protein translation the code is degenerated [51]. The genetic information is delivered to the cell’s integral protein synthesis machinery – the ribosome – in the form of the mRNA transcript. Inside the ribosome, the actual decoding is mediated by tRNAs that serve as adapter molecules through base-pairing of their anticodon with complementary triplet codons in the mRNA [52]. Being charged with their cognate amino acids, tRNAs provide the ribosome with the protein building blocks and decisively determine the incorporation of a particular amino acid in response to defined codons [53,54]. In the early 1960s Chapeville and colleagues were able to prove Cricks adapter hypothesis by incorporating Ala using an Ala-tRNACys that was obtained by reducing Cys-tRNACys [55]. Using E. coli cell-free translation, this demonstrated the possibility to incorporate amino acids in response to a desired codon by employment of selected tRNA species charged with non-cognate substrates. 3.2. Misacylation of tRNAs The term ‘‘misacylation’’ refers to the aminoacylation of a tRNA with a non-cognate amino acid [6,56]. In their natural context, tRNAs are enzymatically aminoacylated with their cognate amino acids by highly specific aminoacyl-tRNA synthetases (aaRS) [57]. Their amino acid specificity is determined by the amino acid binding pocket and for some aaRSs, an editing domain providing hydrolysis of misacylated tRNAs [58]. Since evolution of the aaRS’s specificity was mainly driven by the correct selection between amino acids present in a given system, it is not surprising that a number of nsAAs has been found to bypass this quality control thereby being selected for protein synthesis [4,59]. Nevertheless, many nsAAs bearing desirable characteristics are not readily accepted at the level of the aaRS, as these enzymes ensure the formation of correctly aminoacylated tRNAs with an accuracy of at least 10 000:1 [60]. However, engineering of selected aaRSs made it possible to alter their amino acid substrate specificity [61]. Unfortunately, it has been shown in vitro, that extensive mutagenesis, even though providing the desired alteration of substrate specificity, often impairs the catalytic activity of aaRSs making them rather inefficient compared to their natural ancestors [62–64]. Besides the enzymatic misacylation of tRNAs different groups have contributed to the establishment of an alternative technology leading to aminoacyl-tRNAs (aatRNA) via a synthetic route [65–70]. This straightforward approach offers implementation of a broader range of nsAAs such as larger organic fluorescent dyes as no aaRSs are involved [27,37,39]. Nevertheless, this procedure remains limited by the elongation factor recognition of



eukaryotic factors represents a much more difficult task due to the larger amount of proteins being involved in eukaryotic protein translation, in particular the initiation phase. Therefore, the utilization of elements that provide translation initiation independent of eukaryotic initiation factors such as viral IRES sequences can be a considered a first step towards the development of such systems. 3.4. Stop codon suppression Of the 64 codons arising from the combination of the four nucleobases in triplets, 3 codons are used to terminate the translation of mRNA into protein. This is achieved by interaction with the release or termination factors (RFs). However, certain organisms exhibit an expanded genetic code to incorporate selected amino acids in response to a stop codon [89]. This principle is referred to as stop codon suppression and is mediated by suppressor tRNAs that contain an anticodon complementary to the respective stop codon. Examples comprise selenocysteine being encoded by the opal codon [1] and pyrrolysine by the amber codon [2,3]. Stop codon suppression, in particular amber codon suppression, has emerged to be the most widely used method to incorporate nsAAs in a site-directed manner. Thereby, the desired position can be easily determined by site-directed mutagenesis. Due to a competition of the RFs and the suppressor tRNA for interaction with the stop codon, usually two types of translation products are obtained: the truncated termination product and the full-length suppression product containing the desired nsAA [81]. Several attempts have been made to improve the suppression efficiency, thereby increasing the amount of full-length suppression product. In this context, it is beneficial to incorporate nsAAs in open CFPS as these systems lack the cellular plasma membrane and therefore the translation reaction can be directly influenced by the addition or removal of components. In prokaryotes RF1 specifically recognizes the amber codon (UAG) whereas RF2 recognizes the opal codon (UGA). Termination in response to the ochre codon (UAA) is mediated by overlapping specificity of both RFs [90]. Based on this knowledge, a cell-free RF1-depleted E. coli system has been generated that exhibits significantly increased suppression efficiency with C-terminal termination being mediated by RF2 recognizing the ochre codon [91]. Moreover, heat inactivation of thermo-sensitive RF1 variants [92] as well as RF1 inhibition by aptamers [93] and antibodies [94] have been successfully implemented in CFPS systems. Hong and colleagues just recently demonstrated the incorporation of multiple nsAAs in response to stop codon suppression using extracts from a genetically recoded E. coli strain lacking RF1 [95]. Unfortunately, the approaches mentioned above cannot directly be transferred to eukaryotes, since the eukaryotic release factor 1 (eRF1) is responsible for translation termination in response to all stop codons [96]. In particular, the position of the stop codon to be suppressed plays an important role as it has been found that suppression efficiency is higher the more N-terminal the codon is positioned [37]. Nevertheless, the use of an aptamer in Xenopus oocytes has been shown to slightly increase amber suppression by 3-fold [97]. Additionally, a recent study reported the use of a HEK 293T strain with an engineered eRF1 that increased amber suppression at a single site 17- to 20-fold making this strain a valuable source for future extract preparation [98]. 3.5. Orthogonal tRNA/synthetase pairs The site-directed incorporation of nsAA into proteins based on the utilization of synthetically charged suppressor tRNAs suffers from exhaustion of the aatRNA pool over time. In contrast, the expansion of a given system by an orthogonal aaRS (oRS) that specifically aminoacylates orthogonal suppressor tRNAs (otRNA)

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with nsAAs, provides a continuous replenishment of charged suppressor tRNAs. Orthogonality refers to a lack of cross-reactivity in a given system. In the ideal case, an efficient and site-directed incorporation of nsAAs is achieved when (i) the otRNA is not a substrate for any of the endogenous synthetases and thus is not charged with a sAA, (ii) the oRS does not aminoacylate any endogenous tRNAs with the nsAA and (iii) the oRS exhibits a high specificity for the nsAA and does not accept sAAs as substrates. To meet these requirements, otRNA/oRS pairs are usually imported from phylogenetically distant organisms [99]. For example, archeal wild type PylRS together with its cognate amber suppressor tRNAPyl has been directly employed to incorporate a set of different Pyl analouges in E. coli [100,101] as well as in mammalian cells [102]. Nevertheless, engineering of aaRSs is often required to further enhance orthogonality and efficient aminoacylation of suppressor tRNAs, but also to alter the oRS’s amino acid specificity [61,103,104]. For this purpose, screening methods for the evolution of pairs orthogonal in bacteria and eukaryotes have been established that are based on E. coli [105] and Saccharomyces cerevisiae [106] cells, respectively. Up to now, approximately 100 of such orthogonal translation systems (OTS) have been established [64]. Although, strongly facilitating nsAA incorporation in living cells, their employment in the context of CFPS offers a range of different advantages. As mentioned above engineered oRS often exhibit low activities compared to their natural ancestors especially towards non-cognate suppressor tRNAs [62–64]. To account for this, the open environment of CFPS allows for the addition of defined amounts of purified oRS, otRNA and nsAA to support maintenance of a competitive pool of aatRNAs [107]. Using eukaryotic CFPS in combination with an E. coli derived OTS comprising an amber suppressor tRNA that lacks modified nucleotides, increased Mg2+ concentration have further been found to be beneficial [107]. Furthermore, cytotoxic effects during cell growth, which are often associated with the overexpression of oRSs, otRNAs and the supplemented nsAA itself are circumvented and the preliminary synthesis of the target protein is supported [7]. In this way, even high incorporation efficiencies of poorly soluble nsAAs can be achieved [108]. 3.6. Frame-shift codons The use of frame-shift codons is based on tRNAs with an extended anticodon loop which results in decoding of codons exceeding three nucleobases [109]. In this manner, four-base and even five-base codons have been successfully employed for the incorporation of nsAAs in cell-free protein synthesis [110,111]. In particular, the use of quadruplets derived from rarely employed codons provides an efficient competition with endogenous tRNAs decoding the respective triplet codon [112]. In order to easily discriminate between the frame-shift product and the in-frame translated protein, the codon context is designed in a way that translation is terminated upon triplet decoding [113]. Using synthetically acylated aatRNAs decoding four-base codons Sisido and coworkers achieved a higher incorporation efficiency of nsAAs compared to amber suppression and demonstrated the incorporation of multiple nsAAs in a cell-free E. coli system [111]. Moreover, frame-shift decoding has been transferred to eukaryotic cell-free systems including rabbit reticulocytes and Sf21 extracts [114,115]. Besides the synthetic acylation, it has been shown that four-base decoding tRNAs can also be charged enzymatically [116,117]. 3.7. Orthogonal ribosomes In order to further improve incorporation yields in response to amber suppression, the Chin lab developed orthogonal ribosomes


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that specifically recognize orthogonal mRNA containing a mutant Shine-Delgarno sequence in E. coli [118]. Based on the coexistence of wild-type and orthogonal ribosomes an elegant selection method enabled evolution of an orthogonal ribosome referred to as Ribo-X [119]. Employment of Ribo-X enabled a significantly increased amber suppression rate of orthogonal mRNA resulting from a decreased RF1 termination efficiency [120]. Furthermore, another ribosome mutant, ribosome Q, was found to effectively decode four-base codons. By employment of the two different mutants incorporation of two nsAAs in response to amber suppression and quadruplet decoding was demonstrated in vitro [121]. 3.8. Unnatural base-pairs The use of unnatural base pairs for the generation of unique codons to be employed for the site-directed incorporation on nsAAs represents a promising alternative to the methods described above. Unfortunately, their utilization is impaired by a variety of different problems. Ideally, they must serve as substrates for RNA and DNA polymerases, elongation factors and ribosomes [4]. Moreover, a sufficient orthogonality towards the natural nucleobases is required. The development of unnatural base-pairs including advantages and limitations was recently reviewed by Hirao and Kimoto [122]. Although, it has been demonstrated that unnatural base-pairs can be efficiently suppressed and provide incorporation of nsAAs into proteins in cell-free synthesis, their application still requires further improvements [123,124]. 4. Cell-free applications As explained above, the cell-free approach to incorporate nsAAs benefits from a comparatively high economical efficiency. In particular, this is desired when expensive substrates are used that contain for example stable isotopes or fluorescent dyes. Early on, CFPS was employed in order to investigate the translational flexibility of the protein synthesis apparatus [113,115,125,126]. Moreover, it enables the study of parameters and kinetics influencing tRNA misacylation and amber suppression [63,108,127,128]. Novel orthogonal systems can be characterized in vitro in terms of substrate specificity and efficiency [101]. The final goal of nsAA incorporation is to increase the chemical diversity of proteins (Fig. 1). This may serve to alter protein function [129,130], to introduce new functions [131] or to incorporate biophysical probes or reactive handles for protein conjugation. 4.1. Biophysical probes The significance of a directed incorporation of biophysical probes into proteins is striking, as they allow for highly sensitive detection methods that can be used for quantification purposes as well as structural investigations and many of them do not impede protein function [80,132,133]. CFPS with stable isotope labeled amino acids represents an advantageous method especially in the fields of FT-IR, NMR characterization and MS quantification [134–138]. Cell-free production of stable isotope labeled proteins occurs under optimized conditions with minimized amounts of costly amino acids [139–151]. This is due to the fact that amino acid metabolic pathways are impaired during cell extract preparation and specific inhibitors can be employed which minimizes scrambling [152]. Extracts can also be dialyzed to remove endogenous amino acids in order to decrease dilution. In addition to 13 15 C/ N-labeled amino acids, 19F-substituted nsAAs can be employed for background-free NMR studies [80,127]. 19F is also a valuable biophysical probe because of its chemical shift sensitivity towards the local environment [153]. Furthermore, the use of

heavy atom-substituted amino acids strongly facilitates X-ray crystallographic structure determinations [154,155]. Another important class of biophysical probes comprises fluorescent nsAAs. Fluorescent labeling strategies represent a popular method of investigation due to the high sensitivity of fluorescent probes. Fluorescent nsAAs may also serve as a quick method for the determination of protein yields, eliminating the need for radiolabeling, liquid scintillation counting and exposition on phosphostorage screens [127,128]. The fluorescent probes can be incorporated site-specifically to give information about protein localization, conformation and interactions [156–158]. Incorporation of multiple fluorescent nsAAs enables intramolecular investigations of protein conformation and integrity. Using precharged frameshift-tRNAs, the group of Sisido incorporated two different BODIPY derivatives into calmodulin and was able to observe changes in the FRET signal upon ligand-binding [159]. This report demonstrates the usefulness of small molecular dyes for FRET-studies. In the past, genetic fusions of green fluorescent protein derivatives were applied to monitor conformational changes and protein interactions [160]. The presence of these high-molecular weight (>25kDa) fusion proteins reportedly posed restrictions on protein folding and interactions [161], which can be circumvented with fluorescent nsAAs. Using a mixed approach with amber suppression and four-base decoding, Anderson et al. demonstrated the ability to monitor protein integrity by FRET [162]. In another example, a fluorescent nsAA was used to screen for MMP-9 inhibitors by fluorescence correlation spectroscopy [163]. Moreover, by incorporating a pair of donor/quencher nsAAs into streptavidin, the group of Sisido also demonstrated that this approach yields information about the local protein structure [164]. The binding event of biotin at streptavidin was monitored by incorporated nsAA as well [132,133]. 4.2. Protein conjugation One of the most anticipated fields of applying nsAAs deals with the equipment of proteins with natural and synthetic post-translational modifications (PTMs). Naturally occurring PTMs influence stability, functionality but also localization and interactions of the corresponding modified proteins [165]. However, strong evidence supports a complex crosstalk of different PTM signals but the large diversity makes it difficult to specifically assign certain functions to individual PTMs [166]. In this context, the bottom-up installation of defined PTMs at selected positions within proteins represents a promising approach to decipher nature’s PTM code. Moreover, this approach offers a way to introduced homogenous natural PTM mimics, which may have an important impact on the generation of defined pharmaceutical proteins circumventing the batch to batch variations often accompanied by cell-based methods. Defined PTMs such as mono- and di-glycosylated nsAAs have been incorporated co-translationally into proteins by CFPS [167]. Alternatively, the incorporation of nsAAs with reactive handles also allows for the subsequent post-translational modification of synthesized proteins. For instance, PTM mimics such as phosphotyrosine analogues [168] or truly artificial ones like polyethylenglycol [169] have been demonstrated. In principle, any protein modification is feasible via this approach, as the translational machinery does not regulate the selected posttranslational modification of amino acid side chains. In this manner, the conjugation of biophysical probes [107,170–172], affinity handles [173] and an oriented immobilization [174] can be achieved. Using photoreactive nsAAs, protein interactions can be analyzed for example to investigate protein trafficking [175]. The incorporation of nsAAs with two corresponding reactive groups enables direct conjugation of proteins by the



copper-catalyzed alkyne-azide cycloaddition (CuAAC) [176]. This allows for the generation of antibody-drug-conjugates [177], antibody-display on virus-like-particles [178] and the covalent linkage of single-chain antibody fragments to reporter proteins [179]. In spite of the reported drawbacks of the active copper(I) species in biological environments, the bioorthogonal CuAAC reaction is extremely useful because of its relatively fast kinetics and convincing selectivity even within complex biological environments [180]. Finally different reactive groups for novel bioorthogonal transformations are under investigation bearing great potential with regard to improved selectivity and kinetics under physiological conditions [180]. 5. Conclusion The implementation of nsAA incorporation into CFPS has in various examples been demonstrated to be beneficial thereby providing a promising complementary platform to cell-based approaches. In particular with regard to the characterization of novel incorporation methods, engineered orthogonal tRNA/synthetase pairs and novel methodologies for improved nsAA incorporation efficiency, CFPS offers a straightforward methodology susceptible to high-throughput approaches. It is obvious that more randomized mutant screenings will allow for the generation of aaRS with enhanced activities thus facilitating nsAA incorporation. In this context, eukaryotic cell-free system are particularly promising platforms as mutant library screenings based on eukaryotic cells suffer from a limited throughput. The broad diversity and the multitude of CFPS systems currently available will eventually provide new biological insights gained from the characterization of proteins that are difficult or even impossible to study in living cells such as toxic proteins, proteins containing homogenous PTMs and certain membrane proteins. The ongoing efforts of scientist to enhance translation rates in combination with high-yielding reaction formats are promising to pave the way towards the application of eukaryotic cell-free systems for large-scale protein production. Acknowledgments This work is supported by the German Ministry of Education and Research (BMBF, Nos. 0315942 and 0312039) and the German Research Foundation (DFG Priority Programme 1623). The authors declare that there are no conflicts of interest. References [1] Bock, A., Forchhammer, K., Heider, J. and Baron, C. (1991) Selenoprotein synthesis: an expansion of the genetic code. Trends Biochem. Sci. 16, 463– 467. [2] Hao, B., Gong, W., Ferguson, T.K., James, C.M., Krzycki, J.A. and Chan, M.K. (2002) A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466. [3] Srinivasan, G., James, C.M. and Krzycki, J.A. (2002) Pyrrolysine encoded by UAG in archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462. [4] Magliery, T.J. (2005) Unnatural protein engineering: producing proteins with unnatural amino acids. Med. Chem. Rev. – Online 2, 303–323. [5] Liu, C.C. and Schultz, P.G. (2010) Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444. [6] Budisa, N. (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew. Chem. Int. Ed. 43, 6426–6463. [7] Hong, S.H., Kwon, Y.-C. and Jewett, M.C. (2014) Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis. Front. Chem. 2. [8] Rosenblum, G. and Cooperman, B.S. (2014) Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett. 588, 261–268. [9] Katzen, F., Chang, G. and Kudlicki, W. (2005) The past, present and future of cell-free protein synthesis. Trends Biotechnol. 23, 150–156.

7

[10] Carlson, E.D., Gan, R., Hodgman, C.E. and Jewett, M.C. (2012) Cell-free protein synthesis: applications come of age. Biotechnol. Adv. 30, 1185–1194. [11] Murray, C.J. and Baliga, R. (2013) Cell-free translation of peptides and proteins: from high throughput screening to clinical production. Curr. Opin. Chem. Biol. 17, 420–426. [12] Endo, Y. and Sawasaki, T. (2006) Cell-free expression systems for eukaryotic protein production. Protein Technol. 17, 373–380. [13] Kigawa, T., Yabuki, T., Matsuda, N., Matsuda, T., Nakajima, R., Tanaka, A. and Yokoyama, S. (2004) Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63–68. [14] Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y. and Alakhov, Y.B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164. [15] Fuchs, U., Stiege, W. and Erdmann, V.A. (1997) Ribonucleolytic activities in the Escherichia coli in vitro translation system and in its separate components. FEBS Lett. 414, 362–364. [16] Kim, D.-M. and Swartz, J.R. (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66, 180–188. [17] Kovtun, O., Mureev, S., Jung, W., Kubala, M.H., Johnston, W. and Alexandrov, K. (2011) Leishmania cell-free protein expression system. Methods 55. [18] Uzawa, T., Yamagishi, A. and Oshima, T. (2003) Continuous cell-free protein synthesis directed by messenger DNA and catalyzed by extract of Thermus thermophilus HB27. Biosci. Biotechnol. Biochem. 67, 639–642. [19] Takai, K., Sawasaki, T. and Endo, Y. (2010) Practical cell-free protein synthesis system using purified wheat embryos. Nat. Protoc. 5, 227–238. [20] Madin, K., Sawasaki, T., Ogasawara, T. and Endo, Y. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. 97, 559–564. [21] Buntru, M., Vogel, S., Spiegel, H. and Schillberg, S. (2014) Tobacco BY-2 cellfree lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnol. 14, 37. [22] Ezure, T., Suzuki, T., Shikata, M., Ito, M. and Ando, E. (2010) A cell-free protein synthesis system from insect cells. Methods Mol. Biol. 607. [23] Tarui, H., Imanishi, S. and Hara, T. (2000) A novel cell-free translation/ glycosylation system prepared from insect cells. J. Biosci. Bioeng. 90, 508– 514. [24] Stech, M., Quast, R.B., Sachse, R., Schulze, C., Wüstenhagen, D.A. and Kubick, S. (2014) A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells. PLoS One 9. [25] Hodgman, C.E. and Jewett, M.C. (2013) Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis. Biotechnol. Bioeng. 110, 2643–2654. [26] Pelham, H.R.B. and Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247–256. [27] Brödel, A.K., Sonnabend, A. and Kubick, S. (2014) Cell-free protein expression based on extracts from CHO cells. Biotechnol. Bioeng. 111. [28] McDowell, M.J., Joklik, W.K., Villa-Komaroff, L. and Lodish, H.F. (1972) Translation of reovirus messenger RNAs synthesized in vitro into reovirus polypeptides by several mammalian cell-free extracts. Proc. Natl. Acad. Sci. U.S.A. 69, 2649–2653. [29] Rakotondrafara, A.M. and Hentze, M.W. (2011) An efficient factor-depleted mammalian in vitro translation system. Nat. Protoc. 6, 563–571. [30] Bergamini, G., Preiss, T. and Hentze, M.W. (2000) Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA 6, 1781–1790. [31] Hahn, G.-H. and Kim, D.-M. (2006) Production of milligram quantities of recombinant proteins from PCR-amplified DNAs in a continuous-exchange cell-free protein synthesis system. Anal. Biochem. 355, 151–153. [32] Hoffmann, T., Nemetz, C., Schweizer, R., Mutter, W. and Watzele, M. (2002) High-level cell-free protein expression from PCR-generated DNA templates in: Cell-Free Translation Systems (Spirin, A., Ed.), pp. 203–210, Springer, Berlin Heidelberg. [33] Sawasaki, T., Ogasawara, T., Morishita, R. and Endo, Y. (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. 99, 14652–14657. [34] Sachse, R., Dondapati, S.K., Fenz, S.F., Schmidt, T. and Kubick, S. (2014) Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes. FEBS Lett. 588. [35] Harbers, M. (2014) Wheat germ systems for cell-free protein expression. FEBS Lett. 588, 2762–2773. [36] S. Kubick, M. Gerrits, H. Merk, W. Stiege, V.A. Erdmann, In vitro synthesis of posttranslationally modified membrane proteins, In Current Topics in Membranes Elsevier, 2009. [37] Ezure, T., Nanatani, K., Sato, Y., Suzuki, S., Aizawa, K., Souma, S., Ito, M., Hohsaka, T., von Heijine, G., Utsumi, T., Abe, K., Ando, E. and Uozumi, N. (2014) A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins. PLoS ONE 9, e112874. [38] Brödel, A., Wüstenhagen, D. and Kubick, S. (2015) Cell-free protein synthesis systems derived from cultured mammalian cells in: Structural Proteomics (Owens, R.J., Ed.), pp. 129–140, Springer, New York. [39] Sachse, R., Wüstenhagen, D., Šamalíková, M., Gerrits, M., Bier, F.F. and Kubick, S. (2013) Synthesis of membrane proteins in eukaryotic cell-free systems. Eng. Life Sci. 13, 39–48.


8


[40] Shaklee, P.M., Semrau, S., Malkus, M., Kubick, S., Dogterom, M. and Schmidt, T. (2010) Protein incorporation in giant lipid vesicles under physiological conditions. ChemBioChem 11, 175–179. [41] Zampatis, D.E., Rutz, C., Furkert, J., Schmidt, A., Wüstenhagen, D., Kubick, S., Tsopanoglou, N.E. and Schülein, R. (2012) The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression. FEBS Lett. 586, 2351–2359. [42] Merk, H., Gless, C., Maertens, B., Gerrits, M. and Stiege, W. (2012) Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes. BioTechniques 53, 153–160. [43] Stech, M., Hust, M., Schulze, C., Dübel, S. and Kubick, S. (2014) Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments. Eng. Life Sci. 14, 387–398. [44] Dondapati, S.K., Kreir, M., Quast, R.B., Wüstenhagen, D.A., Bruggemann, A., Fertig, N. and Kubick, S. (2014) Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system. Biosens. Bioelectron. 59, 174–183. [45] Hodgman, C.E. and Jewett, M.C. (2014) Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis. New Biotechnol. 31, 499–505. [46] Brödel, A.K., Sonnabend, A., Roberts, L.O., Stech, M., Wüstenhagen, D.A. and Kubick, S. (2013) IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems. PLoS One 8, e82234. [47] Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S. and Imataka, H. (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46, 348–357. [48] Spirin, A.S. (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol. 22, 538–545. [49] Kobayashi, T., Yukigai, J., Ueda, K., Machida, K., Masutani, M., Nishino, Y., Miyazawa, A. and Imataka, H. (2013) Purification and visualization of encephalomyocarditisvirus synthesized by an in vitro protein expression system derived from mammalian cell extract. Biotechnol. Lett. 35, 309–314. [50] Crick, F.H.C., Barnett, L., Brenner, S. and Watts-Tobin, R.J. (1961) General nature of the genetic code for proteins. Nature 192, 1227–1232. [51] Crick, F.H.C. (1966) Codon—anticodon pairing: the wobble hypothesis. J. Mol. Biol. 19, 548–555. [52] Crick, F.H. (1958) On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163. [53] Balasubramaian, R. and Seetharamulu, P. (1983) A conformational rationale for the wobble behaviour of the first base of the anticodon triplet in tRNA. J. Theor. Biol. 101, 77–86. [54] Agris, P.F. (1991) Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis. Biochimie 73, 1345–1349. [55] Chapeville, F., Lipmann, F., Von Ehrenstein, G., Weisblum, B., Ray, W.J.J. and Benzer, S. (1962) On the role of soluble ribonucleic acid in coding for amino acids. Proc. Natl. Acad. Sci. U.S.A. 48, 1086–1092. [56] Stortchevoi, A.A. (2006) Misacylation of tRNA in prokaryotes: a re-evaluation. Cell. Mol. Life Sci. 63, 820–831. [57] Ibba, M. and Söll, D. (2000) Aminoacyl-tRNA synthesis. Annu. Rev. Biochem. 69, 617–650. [58] Jakubowski, H. and Goldman, E. (1992) Editing of errors in selection of amino acids for protein synthesis. Microbiol. Rev. 56, 412–429. [59] Hendrickson, T.L., de Crécy-Lagard, V. and Schimmel, P. (2004) Incorporation of nonnatural amino acids into proteins. Annu. Rev. Biochem. 73, 147–176. [60] Cusack, S. (1997) Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 7, 881–889. [61] Perona, J.J. and Hadd, A. (2012) Structural diversity and protein engineering of the aminoacyl-tRNA synthetases. Biochemistry (Mosc.) 51, 8705–8729. [62] Nehring, S., Budisa, N. and Wiltschi, B. (2012) Performance analysis of orthogonal pairs designed for an expanded eukaryotic genetic code. PLoS One 7, e31992. [63] Albayrak, C. and Swartz, J.R. (2013) Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair. Biochem. Biophys. Res. Commun. 431, 291– 295. [64] O’Donoghue, P., Ling, J., Wang, Y.-S. and Soll, D. (2013) Upgrading protein synthesis for synthetic biology. Nat. Chem. Biol. 9, 594–598. [65] Robertson, S.A., Ellman, J.A. and Schultz, P.G. (1991) A general and efficient route for chemical aminoacylation of transfer RNAs. J. Am. Chem. Soc. 113, 2722–2729. [66] Hecht, S.M., Alford, B.L., Kuroda, Y. and Kitano, S. (1978) ‘‘Chemical aminoacylation’’ of tRNA’s. J. Biol. Chem. 253, 4517–4520. [67] Payne, R.C., Nichols, B.P. and Hecht, S.M. (1987) Escherichia coli tryptophan synthase: synthesis of catalytically competent alpha subunit in a cell-free system containing preacylated tRNAs. Biochemistry (Mosc.) 26, 3197–3205. [68] Ninomiya, K., Kurita, T., Hohsaka, T. and Sisido, M. (2003) Facile aminoacylation of pdCpA dinucleotide with a nonnatural amino acid in cationic micelle. Chem. Commun. (Camb.), 2242–2243. [69] Baldini, G., Martoglio, B., Schachenmann, A., Zugliani, C. and Brunner, J. (1988) Mischarging Escherichia coli tRNAPhe with L-40 -[3-(trifluoromethyl)3H-diazirin-3-yl]phenylalanine, a photoactivatable analog of phenylalanine. Biochemistry (Mosc.) 27, 7951–7959. [70] Robertson, S.A., Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. and Schultz, P.G. (1989) The use of 50 -phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA. Nucleic Acids Res. 17, 9649–9660.

[71] LaRiviere, F.J., Wolfson, A.D. and Uhlenbeck, O.C. (2001) Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation. Science 294, 165–168. [72] Mittelstaet, J., Konevega, A.L. and Rodnina, M.V. (2013) A kinetic safety gate controlling the delivery of unnatural amino acids to the ribosome. J. Am. Chem. Soc. 135, 17031–17038. [73] Bessho, Y., Hodgson, D.R.W. and Suga, H. (2002) A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme. Nat. Biotechnol. 20, 723–728. [74] Murakami, H., Saito, H. and Suga, H. (2003) A versatile tRNA aminoacylation catalyst based on RNA. Chem. Biol. 10, 655–662. [75] Kourouklis, D., Murakami, H. and Suga, H. (2005) Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation. Eng. Transl. 36, 239–244. [76] Murakami, H., Ohta, A., Goto, Y., Sako, Y. and Suga, H. (2006) Flexizyme as a versatile tRNA acylation catalyst and the application for translation. Nucleic Acids Symp. Ser. 50, 35–36. [77] Passioura, T. and Suga, H. (2014) Flexizymes, their evolutionary history and diverse utilities. Top. Curr. Chem. 344, 331–345. [78] Johnson, J.A., Lu, Y.Y., Van Deventer, J.A. and Tirrell, D.A. (2010) Residuespecific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 14, 774–780. [79] Cohen, G.N. and Munier, R. (1956) Incorporation of structural analogues of amino acids in bacterial proteins. Biochim. Biophys. Acta 21, 592–593. [80] Singh-Blom, A., Hughes, R.A. and Ellington, A.D. (2014) An amino acid depleted cell-free protein synthesis system for the incorporation of noncanonical amino acid analogs into proteins. J. Biotechnol. 178. [81] Stech, M., Brödel, A.K., Quast, R.B., Sachse, R. and Kubick, S. (2013) Cell-free systems: functional modules for synthetic and chemical biology. Adv. Biochem. Eng. Biotechnol. 137. [82] Lenhard, B., Orellana, O., Ibba, M. and Weygand-Durasevic, I. (1999) TRNA recognition and evolution of determinants in seryl-tRNA synthesis. Nucleic Acids Res. 27, 721–729. [83] Kobayashi, T., Nureki, O., Ishitani, R., Yaremchuk, A., Tukalo, M., Cusack, S., Sakamoto, K. and Yokoyama, S. (2003) Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion. Nat. Struct. Biol. 10, 425–432. [84] Wang, L. and Schultz, P.G. (2001) A general approach for the generation of orthogonal tRNAs. Chem. Biol. 8, 883–890. [85] Jackson, R.J., Napthine, S. and Brierly, I. (2001) Development of a tRNAdependent in vitro translation system. RNA 7, 765–773. [86] Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S. and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38. [87] Krishnakumar, R. and Ling, J. (2014) Experimental challenges of sense codon reassignment: an innovative approach to genetic code expansion. FEBS Lett. 588, 383–388. [88] Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K. and Ueda, T. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755. [89] Ambrogelly, A., Palioura, S. and Soll, D. (2007) Natural expansion of the genetic code. Nat. Chem. Biol. 3, 29–35. [90] Scolnick, E., Tompkins, R., Caskey, T. and Nirenberg, M. (1968) Release factors differing in specificity for terminator codons. Proc. Natl. Acad. Sci. 61, 768– 774. [91] Gerrits, M., Strey, J., Claußnitzer, I., von Groll, U., Schäfer, F., Rimmele, M. and Stiege, W. (2007) Cell-free synthesis of defined protein conjugates by sitedirected cotranslational labeling in: Cell-free Expression (Kudlicki, T., Katzen, F. and Bennett, R., Eds.), pp. 166–180, Academic Press, Burlington. [92] Short 3rd, G.F., Golovine, S.Y. and Hecht, S.M. (1999) Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids. Biochemistry (Mosc.) 38, 8808–8819. [93] Szkaradkiewicz, K., Nanninga, M., Nesper-Brock, M., Gerrits, M., Erdmann, V.A. and Sprinzl, M. (2002) RNA aptamers directed against release factor 1 from Thermus thermophilus. FEBS Lett. 514, 90–95. [94] Agafonov, D.E., Huang, Y., Grote, M. and Sprinzl, M. (2005) Efficient suppression of the amber codon in E. coli in vitro translation system. FEBS Lett. 579, 2156–2160. [95] Hong, S.H., Ntai, I., Haimovich, A.D., Kelleher, N.L., Isaacs, F.J. and Jewett, M.C. (2014) Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation. ACS Synth. Biol. 3. [96] Frolova, L., Le Goff, X., Rasmussen, H.H., Cheperegin, S., Drugeon, G., Kress, M., Arman, I., Haenni, A.-L., Celis, J.E., Phllippe, M., Justesen, J. and Kisselev, L. (1994) A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372, 701–703. [97] Shafer, A.M., Kálai, T., Bin Liu, S.Q., Hideg, K. and Voss, J.C. (2004) Site-specific insertion of spin-labeled L-amino acids in xenopus oocytes . Biochemistry (Mosc.) 43, 8470–8482. [98] Schmied, W.H., Elsässer, S.J., Uttamapinant, C. and Chin, J.W. (2014) Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577–15583. [99] Kim, C.H., Axup, J.Y. and Schultz, P.G. (2013) Protein conjugation with genetically encoded unnatural amino acids. Gene TherEnergy 17, 412–419.


R.B. Quast et al. / FEBS Letters xxx (2015) xxx–xxx [100] Kobayashi, T., Yanagisawa, T., Sakamoto, K. and Yokoyama, S. (2009) Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase. J. Mol. Biol. 385, 1352–1360. [101] Polycarpo, C.R., Herring, S., Bérubé, A., Wood, J.L., Söll, D. and Ambrogelly, A. (2006) Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 580, 6695–6700. [102] Mukai, T., Kobayashi, T., Hino, N., Yanagisawa, T., Sakamoto, K. and Yokoyama, S. (2008) Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822. [103] Takimoto, J.K., Adams, K.L., Xiang, Z. and Wang, L. (2009) Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells. Mol. Biosyst. 5, 931–934. [104] Yanagisawa, T., Ishii, R., Fukunaga, R., Kobayashi, T., Sakamoto, K. and Yokoyama, S. (2008) Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for sitespecific protein modification. Chem. Biol. 15, 1187–1197. [105] Santoro, S.W., Wang, L., Herberich, B., King, D.S. and Schultz, P.G. (2002) An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20, 1044–1048. [106] Chin, J.W., Cropp, T.A., Anderson, J.C., Mukherji, M., Zhang, Z. and Schultz, P.G. (2003) An expanded eukaryotic genetic code. Science 301, 964–967. [107] Quast, R.B., Claussnitzer, I., Merk, H., Kubick, S. and Gerrits, M. (2014) Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems. Anal. Biochem. 451. [108] Albayrak, C. and Swartz, J.R. (2013) Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation. Nucleic Acids Res. 41, 5949–5963. [109] Dinman, J.D., Icho, T. and Wickner, R.B. (1991) A 1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein. Proc. Natl. Acad. Sci. 88, 174–178. [110] Hohsaka, T. and Sisido, M. (2000) Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs. Nucleic Acids Symp. Ser. (44), 99–100. [111] Hohsaka, T., Ashizuka, Y., Taira, H., Murakami, H. and Sisido, M. (2001) Incorporation of nonnatural amino acids into proteins by using various fourbase codons in an Escherichia coli in vitro translation system. Biochemistry (Mosc.) 40, 11060–11064. [112] Taira, H., Hohsaka, T. and Sisido, M. (2006) In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Nucleic Acids Res. 34, 1653–1662. [113] Hohsaka, T., Kajihara, D., Ashizuka, Y., Murakami, H. and Sisido, M. (1998) Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems. J. Am. Chem. Soc. 121, 34–40. [114] Hohsaka, T., Fukushima, M. and Sisido, M. (2002) Nonnatural mutagenesis in E. coli and rabbit reticulocyte lysates by using four-base codons. Nucleic Acids Res. Suppl. 2001, 201–202. [115] Taki, M., Tokuda, Y., Ohtsuki, T. and Sisido, M. (2006) Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system. J. Biosci. Bioeng. 102, 511–517. [116] Anderson, J.C. and Schultz, P.G. (2003) Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. Biochemistry (Mosc.) 42, 9598–9608. [117] O’Donoghue, P., Prat, L., Heinemann, I.U., Ling, J., Odoi, K., Liu, W.R. and Söll, D. (2012) Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion. FEBS Lett. 586, 3931–3937. [118] Rackham, O. and Chin, J.W. (2005) A network of orthogonal ribosome RNA pairs. Nat. Chem. Biol. 1, 159–166. [119] Wang, K., Neumann, H., Peak-Chew, S.Y. and Chin, J.W. (2007) Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat. Biotechnol. 25, 770–777. [120] Barrett, O.P.T. and Chin, J.W. (2010) Evolved orthogonal ribosome purification for in vitro characterization. Nucleic Acids Res. 38 (8), 2682– 2691, http://dx.doi.org/10.1093/nar/gkq120. [121] Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. and Chin, J.W. (2010) Encoding multiple unnatural amino acids via evolution of a quadrupletdecoding ribosome. Nature 464, 441–444. [122] Hirao, I. and Kimoto, M. (2012) Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma. Proc. Jpn. Acad. B Phys. Biol. Sci. 88, 345–367. [123] Bain, J.D., Switzer, C., Chamberlin, R. and Bennert, S.A. (1992) Ribosomemediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code. Nature 356, 537–539. [124] Hirao, I., Ohtsuki, T., Fujiwara, T., Mitsui, T., Yokogawa, T., Okuni, T., Nakayama, H., Takio, K., Yabuki, T., Kigawa, T., Kodama, K., Yokogawa, T., Nishikawa, K. and Yokoyama, S. (2002) An unnatural base pair for incorporating amino acid analogs into proteins. Nat. Biotechnol. 20, 177–182. [125] Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. and Schultz, P.G. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188.

9

[126] Cornish, V.W., Benson, D.R., Altenbach, C.A., Hideg, K., Hubbell, W.L. and Schultz, P.G. (1994) Site-specific incorporation of biophysical probes into proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 2910–2914. [127] Loscha, K.V., Herlt, A.J., Qi, R., Huber, T., Ozawa, K. and Otting, G. (2012) Multiple-site labeling of proteins with unnatural amino acids. Angew. Chem. Int. Ed. 51, 2243–2246. [128] Ozawa, K., Loscha, K.V., Kuppan, K.V., Loh, C.T., Dixon, N.E. and Otting, G. (2012) High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites. Biochem. Biophys. Res. Commun. 418, 652–656. [129] Bae, J.H., Rubini, M., Jung, G., Wiegand, G., Seifert, M.H.J., Azim, M.K., Kim, J.S., Zumbusch, A., Holak, T.A., Moroder, L., Huber, R. and Budisa, N. (2003) Expansion of the genetic code enables design of a novel ‘‘gold’’ class of green fluorescent proteins. J. Mol. Biol. 328, 1071–1081. [130] Welsh, J.P., Patel, K.G., Manthiram, K. and Swartz, J.R. (2009) Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence. Biochem. Biophys. Res. Commun. 389, 563–568. [131] Hyster, T.K., Knörr, L., Ward, T.R. and Rovis, T. (2012) Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C–H activation. Science 338, 500–503. [132] Murakami, H., Hohsaka, T., Ashizuka, Y., Hashimoto, K. and Sisido, M. (2000) Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin. Biomacromolecules 1, 118–125. [133] Taki, M., Hohsaka, T., Murakami, H., Taira, K. and Sisido, M. (2001) A nonnatural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett. 507, 35–38. [134] Maslennikov, I. and Choe, S. (2013) Advances in NMR structures of integral membrane proteins. Curr. Opin. Struct. Biol. 23, 555–562. [135] Sobhanifar, S., Reckel, S., Junge, F., Schwarz, D., Kai, L., Karbyshev, M., Lohr, F., Bernhard, F. and Dotsch, V. (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J. Biomol. NMR 46. [136] Sonar, S., Lee, C.-P., Coleman, M., Patel, N., Liu, X., Marti, T., Khorana, H.G., RajBhandary, U.L. and Rothschild, K.J. (1994) Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin. Nat. Struct. Mol. Biol. 1, 512– 517. [137] Jones, D.H., Cellitti, S.E., Hao, X., Zhang, Q., Jahnz, M., Summerer, D., Schultz, P.G., Uno, T. and Geierstanger, B.H. (2010) Site-specific labeling of proteins with NMR-active unnatural amino acids. J. Biomol. NMR 46. [138] Singh, S., Kirchner, M., Steen, J.A. and Steen, H. (2012) A practical guide to the FLEXIQuant method. Methods Mol. Biol. 893. [139] Kigawa, T., Muto, Y. and Yokoyama, S. (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6, 129–134. [140] Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T. and Yokoyama, S. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19. [141] Torizawa, T., Shimizu, M., Taoka, M., Miyano, H. and Kainosho, M. (2004) Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol. J. Biomol. NMR 30, 311–325. [142] Klammt, C., Lohr, F., Schafer, B., Haase, W., Dotsch, V., Ruterjans, H., Glaubitz, C. and Bernhard, F. (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. FEBS 271, 568–580. [143] Kainosho, M., Torizawa, T., Iwashita, Y., Terauchi, T., Mei Ono, A. and Guntert, P. (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440, 52–57. [144] Maslennikov, I., Klammt, C., Hwang, E., Kefala, G., Okamura, M., Esquivies, L., Mors, K., Glaubitz, C., Kwiatkowski, W., Jeon, Y.H. and Choe, S. (2010) Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis. Proc. Natl. Acad. Sci. U.S.A. 107, 10902–10907. [145] Nguyen, T.H.D., Ozawa, K., Stanton-Cook, M., Barrow, R., Huber, T. and Otting, G. (2011) Generation of pseudocontact shifts in protein NMR spectra with a genetically encoded cobalt(II)-binding amino acid. Angew. Chem. Int. Ed. 50, 692–694. [146] Reckel, S., Gottstein, D., Stehle, J., Lohr, F., Verhoefen, M.-K., Takeda, M., Silvers, R., Kainosho, M., Glaubitz, C., Wachtveitl, J., Bernhard, F., Schwalbe, H., Guntert, P. and Dotsch, V. (2011) Solution NMR structure of proteorhodopsin. Angew. Chem. Int. Ed Engl. 50, 11942–11946. [147] Michel, E. and Wuthrich, K. (2012) Cell-free expression of disulfidecontaining eukaryotic proteins for structural biology. FEBS J. 279, 3176– 3184. [148] Klammt, C., Maslennikov, I., Bayrhuber, M., Eichmann, C., Vajpai, N., Chiu, E.J.C., Blain, K.Y., Esquivies, L., Kwon, J.H.J., Balana, B., Pieper, U., Sali, A., Slesinger, P.A., Kwiatkowski, W., Riek, R. and Choe, S. (2012) Facile backbone structure determination of human membrane proteins by NMR spectroscopy. Nat. Methods 9, 834–839. [149] Michel, E. and Wuthrich, K. (2012) High-yield Escherichia coli-based cell-free expression of human proteins. J. Biomol. NMR 53. [150] Michel, E., Skrisovska, L., Wuthrich, K. and Allain, F.H.-T. (2013) Amino acidselective segmental isotope labeling of multidomain proteins for structural biology. Chembiochem. Eur. J. Chem. Biol. 14, 457–466. [151] Hagn, F., Etzkorn, M., Raschle, T. and Wagner, G. (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J. Am. Chem. Soc. 135, 1919–1925.


10


[152] Su, X.-C., Loh, C.-T., Qi, R. and Otting, G. (2011) Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enzymes for selective 15N-labelling and production of perdeuterated proteins in H2O. J. Biomol. NMR 50. [153] Neerathilingam, M., Greene, L.H., Colebrooke, S.A., Campbell, I.D. and Staunton, D. (2005) Quantitation of protein expression in a cell-free system: efficient detection of yields and 19F NMR to identify folded protein. J. Biomol. NMR 31, 11–19. [154] Kiga, D., Sakamoto, K., Kodama, K., Kigawa, T., Matsuda, T., Yabuki, T., Shirouzu, M., Harada, Y., Nakayama, H., Takio, K., Hasegawa, Y., Endo, Y., Hirao, I. and Yokoyama, S. (2002) An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cellfree system. Proc. Natl. Acad. Sci. U.S.A. 99, 9715–9720. [155] Kigawa, T., Yamaguchi-Nunokawa, E., Kodama, K., Matsuda, T., Yabuki, T., Matsuda, N., Ishitani, R., Nureki, O. and Yokoyama, S. (2002) Selenomethionine incorporation into a protein by cell-free synthesis. J. Struct. Funct. Genomics 2. [156] Ohtsuka, T., Neki, S., Kanai, T., Akiyoshi, K., Nomura, S.M. and Ohtsuki, T. (2011) Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes. Anal. Biochem. 418. [157] Abe, R., Jeong, H.-J., Arakawa, D., Dong, J., Ohashi, H., Kaigome, R., Saiki, F., Yamane, K., Takagi, H. and Ueda, H. (2014) Ultra Q-bodies: quench-based antibody probes that utilize dye–dye interactions with enhanced antigendependent fluorescence. Sci. Rep. 4. [158] Ugwumba, I.N., Ozawa, K., de la Cruz, L., Xu, Z.-Q., Herlt, A.J., Hadler, K.S., Coppin, C., Brown, S.E., Schenk, G., Oakeshott, J.G. and Otting, G. (2011) Using a genetically encoded fluorescent amino acid as a site-specific probe to detect binding of low-molecular-weight compounds. Assay Drug Dev. Technol. 9, 50–57. [159] Kajihara, D., Abe, R., Iijima, I., Komiyama, C., Sisido, M. and Hohsaka, T. (2006) FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat. Methods 3, 923–929. [160] Giepmans, B.N.G., Adams, S.R., Ellisman, M.H. and Tsien, R.Y. (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217–224. [161] Zhang, J., Campbell, R.E., Ting, A.Y. and Tsien, R.Y. (2002) Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918. [162] Anderson 3rd, R.D., Zhou, J. and Hecht, S.M. (2002) Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase. J. Am. Chem. Soc. 124, 9674–9675. [163] Nakata, H., Ohtsuki, T. and Sisido, M. (2009) A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates. Anal. Biochem. 390, 121–125. [164] Taki, M., Hohsaka, T., Murakami, H., Taira, K. and Sisido, M. (2002) Positionspecific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs. J. Am. Chem. Soc. 124, 14586–14590. [165] Knorre, D.G., Kudryashova, N.V. and Godovikova, T.S. (2009) Chemical and functional aspects of posttranslational modification of proteins. Acta Nat. 1. [166] Venne, A.S., Kollipara, L. and Zahedi, R.P. (2014) The next level of complexity: crosstalk of posttranslational modifications. Proteomics 14, 513–524. [167] Fahmi, N.E., Dedkova, L., Wang, B., Golovine, S. and Hecht, S.M. (2007) Sitespecific incorporation of glycosylated serine and tyrosine derivatives into proteins. J. Am. Chem. Soc. 129, 3586–3597. [168] Serwa, R., Wilkening, I., Del Signore, G., Mühlberg, M., Claußnitzer, I., Weise, C., Gerrits, M. and Hackenberger, C.P.R. (2009) Chemoselective Staudingerphosphite reaction of azides for the phosphorylation of proteins. Angew. Chem. Int. Ed. 48, 8234–8239. [169] Serwa, R., Majkut, P., Horstmann, B., Swiecicki, J.-M., Gerrits, M., Krause, E. and Hackenberger, C.P.R. (2010) Site-specific PEGylation of proteins by a Staudinger-phosphite reaction. Chem. Sci. 1, 596–602. [170] Abe, M., Ohno, S., Yokogawa, T., Nakanishi, T., Arisaka, F., Hosoya, T., Hiramatsu, T., Suzuki, M., Ogasawara, T., Sawasaki, T., Nishikawa, K., Kitamura, M., Hori, H. and Endo, Y. (2007) Detection of structural changes

[171]

[172]

[173]

[174]

[175]

[176]

[177]

[178]

[179]

[180] [181]

[182]

[183]

[184]

[185]

[186]

[187]

[188]

in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing. Proteins 67, 643–652. Goerke, A.R. and Swartz, J.R. (2009) High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids. Biotechnol. Bioeng. 102, 400–416. Uzagare, M.C., Claussnitzer, I., Gerrits, M. and Bannwarth, W. (2012) Site specific chemoselective labelling of proteins with robust and highly sensitive Ru(II) bathophenanthroline complexes. Org. Biomol. Chem. 10, 2223–2226. Ohno, S., Matsui, M., Yokogawa, T., Nakamura, M., Hosoya, T., Hiramatsu, T., Suzuki, M., Hayashi, N. and Nishikawa, K. (2007) Site-selective posttranslational modification of proteins using an unnatural amino acid, 3azidotyrosine. J. Biochem. (Tokyo) 141, 335–343. Smith, M.T., Wu, J.C., Varner, C.T. and Bundy, B.C. (2013) Enhanced protein stability through minimally invasive, direct, covalent, and site-specific immobilization. Biotechnol. Prog. 29, 247–254. Kanamori, T., Nishikawa, S., Shin, I., Schultz, P.G. and Endo, T. (1997) Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking. Proc. Natl. Acad. Sci. U.S.A. 94, 485–490. Bundy, B.C. and Swartz, J.R. (2010) Site-specific incorporation of ppropargyloxyphenylalanine in a cell-free environment for direct proteinprotein click conjugation. Bioconjug. Chem. 21, 255–263. Zimmerman, E.S., Heibeck, T.H., Gill, A., Li, X., Murray, C.J., Madlansacay, M.R., Tran, C., Uter, N.T., Yin, G., Rivers, P.J., Yam, A.Y., Wang, W.D., Steiner, A.R., Bajad, S.U., Penta, K., Yang, W., Hallam, T.J., Thanos, C.D. and Sato, A.K. (2014) Production of site-specific antibody-drug conjugates using optimized nonnatural amino acids in a cell-free expression system. Bioconjug. Chem. 25, 351–361. Patel, K.G. and Swartz, J.R. (2011) Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry. Bioconjug. Chem. 22, 376–387. Patel, K.G., Ng, P.P., Kuo, C.-C., Levy, S., Levy, R. and Swartz, J.R. (2009) Cellfree production of Gaussia princeps luciferase – antibody fragment bioconjugates for ex vivo detection of tumor cells. Biochem. Biophys. Res. Commun. 390, 971–976. Lang, K. and Chin, J.W. (2014) Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16–20. Steward, L.E., Collins, C.S., Gilmore, M.A., Carlson, J.E., Ross, J.B.A. and Chamberlin, A.R. (1997) In vitro site-specific incorporation of fluorescent probes into b-galactosidase. J. Am. Chem. Soc. 119, 6–11. Mukai, T., Yanagisawa, T., Ohtake, K., Wakamori, M., Adachi, J., Hino, N., Sato, A., Kobayashi, T., Hayashi, A., Shirouzu, M., Umehara, T., Yokoyama, S. and Sakamoto, K. (2011) Genetic-code evolution for protein synthesis with nonnatural amino acids. Biochem. Biophys. Res. Commun. 411, 757–761. Kodama, K., Nakayama, H., Sakamoto, K., Fukuzawa, S., Kigawa, T., Yabuki, T., Kitabatake, M., Takio, K. and Yokoyama, S. (2010) Site-specific incorporation of 4-iodo-L-phenylalanine through opal suppression. J. Biochem. (Tokyo) 148, 179–187. Wang, B., Brown, K.C., Lodder, M., Craik, C.S. and Hecht, S.M. (2002) Chemically mediated site-specific proteolysis. Alteration of protein–protein interaction. Biochemistry (Mosc.) 41, 2805–2813. Li, S., Millward, S. and Roberts, R. (2002) In vitro selection of mRNA display libraries containing an unnatural amino acid. J. Am. Chem. Soc. 124, 9972– 9973. Watanabe, T., Muranaka, N., Iijima, I. and Hohsaka, T. (2007) Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cellfree translation system. Biochem. Biophys. Res. Commun. 361, 794–799. Ozawa, K., Headlam, M.J., Mouradov, D., Watt, S.J., Beck, J.L., Rodgers, K.J., Dean, R.T., Huber, T., Otting, G. and Dixon, N.E. (2005) Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins. FEBS J. 272, 3162–3171. Iijima, I. and Hohsaka, T. (2009) Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching. Chembiochem. Eur. J. Chem. Biol. 10.


Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.

Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids.

Synthesis and Incorporation of Unnatural Amino Acids To Probe and Optimize Protein Bioconjugations.

Genetic incorporation of recycled unnatural amino acids.

Cotranslational amino-terminal processing.

Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins.

Enzymatic synthesis of chiral γ-amino acids using ω-transaminase.

Intracellular amino acids and protein synthesis in Hela cells.

Effect of ethanol intake on the incorporation of labelled amino acids into liver protein.

Incorporation of specific amino acids as markers of cell differentiation.

Incorporation of labelled amino acids into liver protein after acute ethanol administration.

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids.

Stereoselective synthesis of unsaturated α-amino acids.

Bacterial synthesis of D-amino acids.

Predicting Protein-Protein Interaction Sites Using Sequence Descriptors and Site Propensity of Neighboring Amino Acids.

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

Editing of errors in selection of amino acids for protein synthesis.

The effect of L-3,4-dihydroxyphenylalanine (L-dopa) on the incorporation of radioactive amino acids into rat brain protein.

A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.

Source of amino acids for tRNA acylation. Implications for measurement of protein synthesis.

[Studies using labeled urea on lactating ruminants. 4. Incorporation of 15N-urea into the basic amino acids of goat milk].