Báo cáo khoa học: Optimization of an Escherichia coli system for cell-free synthesis of selectively 15N-labelled proteins for rapid analysis by NMR spectroscopy pdf

Optimization of an Escherichia coli system for cell-free synthesis of selectively 15 N-labelled proteins for rapid analysis by NMR spectroscopy Kiyoshi Ozawa, Madeleine J. Headlam, Patrick M. Schaeffer, Blair R. Henderson, Nicholas E. Dixon and Gottfried Otting Research School of Chemistry, Australian National University, Canberra, Australia Cell-free protein synthesis offers rapid access to proteins that are selectively labelled w ith [ 15 N]amino acids and s uitable for analysis by NMR spectroscopy without chromatographic purification. A system b ased on an Escherichia c oli cell ex- tract was optimized with regard to protein y ield and m inimal usage of 15 N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of u p to 1.8 mg of human cyclophilin A per m L of reaction m edium were obtained b y expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage k p R and p L promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15 N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentri- fugation. Cross-peaks f rom metabolic by-products were evident in the 15 N-HSQC spectra of 13 of the samples. All metabolites were found to be s mall m olecules that could be separated readily from the labelled p roteins b y dialysis. No significant transamination activity was observed except for [ 15 N]Asp, where an enzyme in the cell extract efficiently converted Asp fi Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase a ppeared to be generally reduced in the cell-free expression system. Keywords: cell-free protein s ynthesis; human cyclophilin A; 15 N-HSQC spectrum; NMR; selective stable isotope labe- ling. High-yield, cell-free protein synthesis systems are now available that allow efficient high-throughput production of protein samples for structural genomics and other applications [1–7]. They are also useful for the prod uction of toxic p roteins that interfere with cell r eplication. An attractive application for this method is in production of selectively isotope-labelled samples, as in vitro expression uses much smaller volumes and therefore requires corres- pondingly smaller quantities of expensive isotope-labelled amino acids than conventional in vivo systems. This has been exploited both for the production of stable-isotope labelled proteins for NMR spectroscopy [2,4,7–11], as well as for the incorporation of amino acid analogues [12] such as selenomethionine [13] and 3-iodo- L -tyrosine [14] for X-ray crystallography. Proteinyieldsofupto6mgÆmL )1 have been re ported using expression systems based on Escheri chia coli extracts [2,10,15,16]. At these yields, protein concentrations are sufficiently high t o allow the recording of NMR spectra without further concentration of the reaction medium. In particular, 15 N-HSQC spectra of selectively 15 N-labelled proteins can be recorded without purification of the protein, because only signals from 15 N-labelled a mide groups are detected [16]. 15 N-HSQC spectra present well-resolved, highly sensitive fingerprint information that is uniqu ely suited to assess the NMR properties o f a protein prior to structure determination [17,18]. The p resent study focussed on optimization of a cell-free E. coli expression system with regard to residue-selective 15 N-labelling of proteins. The expression system has been shown previously to be suitable for direct NMR analysis o f the reaction mixture, resulting in minimal sample handling [16]. H owever, t he NMR spectroscopic a nalysis can be complicated by the formation of undesired b y-products which originate from metabolic reactions of the 15 N-labelled amino acids due to the presence of a broad range of enzymes in the E. coli cell extract used in these reactions [16]. While the amino groups from unincorporated [ 15 N]amino acids do not give rise to cross-peaks in 15 N-HSQC spectra due to rapid proton exchange with the water, some of the by-products seem to engage the labelled amino groups in amide bonds. Here we present a spectral catalogue of metabolic by- products obtained for amino acids with [ 15 N]amino[aN] groups to provide a reference for the NMR analysis of Correspondence to G. Otting, Australian National University, Research School of Chemistry, Canberra, ACT 0200, Australia. Fax: +61 2 61250750, Tel.: +61 2 61256507, E-mail: gottfried.otting@anu.edu.au Abbreviations: aaRS, amino-acyl tRNA synthetase; hCypA, human cyclophilin A; RNAP, RNA polymerase; s-CYPA, synthetic gene encoding hCypA. (Received 2 4 June 200 4, revised 9 August 2004, accepted 27 August 2004) Eur. J. Biochem. 271, 4084–4093 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04346.x 15 N-labelled protein samples produced in high-throughput mode without further purification or concentration steps. In addition, the following issues were addressed: (a) how do yields c ompare, when transc ription is performed by T7 RNA polymerase from a T7 promoter or by E. coli RNA polymerase from tandem phage k p R and p L promoters; (b) does c ross-labelling among different amino acids occur due to amino- or amido-transferase activity and, if so, can it be s uppressed; (c) which amino acids are prone to the formation of amide-containing by-products; (d) are all by- products sufficiently small to be separated from the pr otein product by dialysis or ultrafiltration; (e) what are the minimum concentrations of labelled amino acids required for good protein y ields and (f) w hich buffer c an be used to replace the large amount of glutamate present in the original medium d escribed b y Y okoyama a nd coworkers [2,15] and also in the buffer of a commercial rapid translation system [11], to enable selective labelling with [ 15 N]Glu? Materials and methods Materials L -[U- 15 N]Arginine.HCl, L -[ 15 N]aspartic acid, L -[U- 15 N]aspa- ragine.H 2 O, L -[ 15 N]cysteine, L -[ 15 N]glutamic acid, L -[ 15 N]glut amine [aN], [ 15 N]glycine, L -[ 15 N]histidine[aN]. HCl, L -[ 15 N]methionine, L -[ 15 N]isoleucine, L -[ 15 N]phenyl- alanine, L -[ 15 N]serine, L -[U- 15 N]tryptophan, L -[ 15 N]tyrosine and L -[ 15 N]valine were purchased from Cambridge I sotope Laboratories (Andover, MA, USA). L -[ 15 N]Alanine, L -[ 15 N]leucine and L -[ 15 N]threonine were obtained from Spectra Stable Isotopes (Columbia, MD, USA) and L -[ 15 N]lysine[aN].2HCl was from Euriso-top (Saint-Aubin, France). Oligonucleotides were purchased from Auspep (Parkville, Australia). Vent DNA polymerase and RNase inhibitor were from Promega, and creatin e kinase and E. coli total tRNA were from Roche. E. coli RNA poly- merase (RNAP) holoenzyme was purified as described previously [16]. Spectra/Por 2 d ialysis tubing was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). E. coli strains A19 [16], BL21(DE3)/pLysS [19] and BL21(DE3)recA [20] were as described previously. The plasmid pKE874 [16] was used for production of the E. coli peptidyl-prolyl cis-trans isomerase PpiB under the control of tandem phage k p R and p L promoters. Plasmid pBH964 was used for cell-free expression of a synthetic gene (s-CYPA) that encodes human cyclophilin A (hCypA) under control of the phage T7 promoter in vector pETMCSI [21]. hCypA was p roduced in vivo in strain BL21(DE3)/pLysS/pBH964 as a standard for comparison with protein production using the cell-free system. The co nstruction of the s-CYPA gene and plasmid pBH964, as well as the procedure for purification of hCypA, are described in detail in the Supplementary material. Plasmid pKO1166 contains phage T7 gene 1 (which encodes T7 R NAP) under the transcriptional control of t he phage k p L promoter in vector pMA200U [22]. T7 RNAP was produced in vivo using strain BL21(DE3) recA/ pKO1166. Construction of pKO1166, and the production and purification of the protein are also described in the Supplementary material. Characterization of proteins Protein purity was assessed from SDS/PAGE gels that were stained with Coomassie blue. Except where specified, protein concentrations were determined by the method of Bradford [23] using bovine serum albumin as the standard. The molecular m asses of purified proteins were confirmed by ES I mass spectrometry, using a VG Quattro II mass spectrometer (VG Biotech, Altrincham, UK). The proteins were extensively dialyzed into 0.1% (v/v) formic acid prior to mass spectrometric analysis. Cell-free protein synthesis The K m values of the 20 aminoacyl-tRNA synthetases (aaRS) for each cognate amino a cid, from E. coli strains where possible, were compiled (using the website http:// brenda.bc.uni-koeln.de/) and the aaRSs were categorized into three major groups (Table 1). Groups I–III comprise aaRS with K m values for the appropriate amino acid <10 l M , between 10 and 50 l M , a nd between 50 and 500 l M , respectively. Based on these K m values and the frequency of occurrence of each amino acid in the primary sequence of hCypA, the concentrations of [ 15 N]amino acids chosen for hCypA labelling were 50 l M for [ 15 N]Trp and [ 15 N]Tyr, 150 l M for [ 15 N]Ile, [ 15 N]Thr and [ 15 N]His, 0.35 m M for remaining Group II [ 15 N]amino acids, and 1m M for those in Group III. The S30 cell extract from E. coli strain A19 was prepared by the p rocedure of Pratt [24], followed by c once ntration with polyethylene glycol 8000 as described by Kigawa et al.[2]. In vitro synthesis of PpiB, using the k-promoter vector pKE874 and E. coli RNAP holoenzyme, was essentially as described previously [16]. For in vitro protein synthesis of hCypA, the inner chamber reaction mixtures (total volume 0.7 m L) contained 55 m M HEPES/KOH (pH 7.5), 1.7 m M dithiothreitol, 1.2 m M ATP, 0.8 m M Table 1. Classification of the 20 aminoacyl-tRNA synthetases by K m values. Data compiled from http://brenda.bc.uni-koeln.de/. All values are for E. coli unless noted o th erwise. Enzyme K m (m M ) Group Enzyme K m (m M ) group Group ThrRS a  0.002 I LeuRS  0.05 III TrpRS b ¼ 0.005 I GluRS  0.05 III IleRS  0.005 I AspRS  0.06 III TyrRS  0.008 I SerRS ¼ 0.07 III HisRS  0.008 I MetRS  0.08 III ArgRS ¼ 0.011 II ValRS  0.10 III CysRS c ¼ 0.013 II GlnRS  0.15 III PheRS d  0.028 II GlyRS  0.16 III AsnRS  0.032 II AlaRS  0.34 III ProRS e – II LysRS f – III a Value for Saccharomyces carlsbergensis, no data available for E. coli; b value for Lupinus luteus and bovine, no data available for E. coli; c value for Paracoccus denitrificans, no data available for E. coli. d value for Bacillus subtilis, no data available for E. coli; e no data available, assigned to group II based on side-chain rigidity; f no data available, assigned to group III based on simi- larity with Glu and Met. Ó FEBS 2004 Cell-free synthesis of 15 N-labelled proteins (Eur. J. Biochem. 271) 4085 each of CTP, GTP and UTP, 0.64 m M 3¢,5¢-cyclic AMP, 68 l M folinic acid, 27.5 m M ammonium acetate, 208 m M potassium glutamate, 80 m M creatine phosphate, 250 lgÆmL )1 creatine kinase, a [ 15 N]aminoacid(atthe concentration given above), 1 m M each of 19 unlabelled L -amino ac ids, 1 5 m M magnesium acetate, 175 lgÆmL )1 E. coli total t RNA, 0.05% (w/v) N aN 3 ,168lLof concentrated S30 extract (containing 5.2 mg of total protein), 16 lgÆmL )1 of supercoiled plasmid DNA (pBH964, as described above), 150 U of RNase inhibitor and 93 lgÆmL )1 of T7 RNAP. For labelling with [ 15 N]Glu, ammonium or potassium acetate (200 m M ) was used instead of potassium glutamate (208 m M ). The inner chamber reaction mixtures were dialyzed in Spectra/Por 2 tubing with a nominal size cutoff of 12–14 kDa for 10 or 12 h at 37 °C with gentle shaking against the outer chamber solution (14 mL) that was changed after 3 a nd 6 h. The inner chamber assembly and the outer chamber b uffer were housed within a 50-mL polypropylene tube. T he outer chamber solution had the same composition as the inner chamber reaction mixture, except that S30 extract, tRNA, plasmid DNA, T7 RNAP, creatine kinase and RNase inhibitor were omitted, and the concentration of magnesium acetate was increased to 19.3 m M . For SDS/PAGE analysis, in vitro reaction samples were diluted two-fold with gel loading mix containing 2% (w/v) SDS and heated for 2 min at 90 °C. The reaction mixtures containing hCypA were clarified by ultracentrifu- gation (100 000 g,4h)andstoredat4°C. NMR spectroscopy All NMR spectra were recorded at 25 °CusingaVarian INOVA 600 MHz NMR spectrometer equipped w ith a probe operating at room temperature. 15 N-HSQC spectra were recorded with 5 mm sample tubes using t 1max ¼ 32 ms, t 2max ¼ 102 ms a nd total recording t imes of 17–24 h. NMR measurements were made using the super- natant from the clarified r eaction mixtures after addition of 10% (v/v) D 2 O to provide a lock signal. In addition, spectra were recorded after dialysis of the samples overnight at 4 °C in Spectra/Por 2 tubing, against buffer comprising 1 0 m M sodium phosphate (pH 6.5), 100 m M NaNO 3 ,5m M dithiothreitol and 5 0 l M NaN 3 . The dialyzed samples were concentrated to a final v olume o f about 0.6 mL using Millipore Ultra-4 centrifugal filters (MWCO 10 000) and D 2 O was added to a final concentration of 10% (v/v) before NMR measurement. Results Cell-free protein synthesis enhanced by T7 RNA polymerase Prior to the preparation of 15 N-labelled samples of hCypA and analysis by NMR spectroscopy, the performance of the cell-free expression system was explored with respect to a number of parameters. We first examined the yields obtained when transcription is conducted by T7 RNAP from the T7 promoter rather than by E. coli RNAP holoenzyme from phage k promoters as in our previous work [16]. Even with promoters recognized by E. coli RNAP, supplementation of the S 30 extract with RNAP i s important for good protein yields [16]. When using T 7 promoters, addition of T7 RNAP was critically required. T7 RNAP, when purchased from commercial sources, is the most expensive component of our c ell-free system. T7 RNAP is, however, relatively easy to produce and purify, as it is a much smaller and simpler p rotein than E. coli RNAP holoenzyme, which is composed of five different subunits (a 2 bb¢xr) [25]. We adapted published methods [26] to develop a simple procedure for the isolation of T7 RNAP from a strain containing a plasmid that directed overproduction of the protein under control of the heat- inducible k p L promoter. Up to 25 mg of pure T7 RNAP could be obtained within a few days from 5 g of cells. This is sufficient for many hundreds of in vitro reactions. Our initial attempts to purify T7 RNAP were hindered by proteolytic cleavage. This was controlled by use of an ompT – E. coli host strain [BL21(DE3)recA], limitation of thetimeculturesweretreatedat42°C during i nduction of synthesis of T7 RNAP to 30 min, followed by treatment for 2 h at 40 °C [ 26], and use of a-toluenesulphonyl fluoride in the buffer during cell lysis. E. coli PpiB [16] was p roduced equally well in vitro when using either T7 or k promoters (data not shown). Corre- spondingly similar y ields were expected f or hCypA, as PpiB and hCypA are f unctional homologues with similar three- dimensional structures and amino acid compositions, and the c odon usage of the CYPA gene had been adjusted for the E. coli expression system by construction of an artificial gene. The protein yields obtained in vitro for hCypA with the T7 promoter system were about 1.5–1.8 mgÆmL )1 of cell-free reaction medium, and were indeed closely c ompar- able to those obtained for PpiB with either promoter system (Fig. 1 , l ane 8 and Fig. 2, lane 1 ). With transcription under the control of the k promoters, however, cell-free synthesis of hCypA was below the detection limit of SDS/PAGE with Coomassie blue staining, even though the same plasmid produced excellent yields in vivo (data not shown). More- over, PpiB was always produced in vitro as a fully soluble protein, whereas a portion (15–20%) of hCypA was invariably found in the insoluble fraction. This is probably due to the pI of hCypA being close to the pH of the reaction mixture (pH 7.5); the pI value of PpiB is about one unit lower. In order t o achieve maximal y ields in the ce ll-free system, smaller mass amounts of T7 RNAP were required than E. coli RNAP, w hen t he same proteins were produced under control o f the T7 and k promoters, respectively (data not shown). A further advantage of the T7 system lies in i ts better tolerance with respect to temperature changes: lowering the reaction temperature from 37 to 30 °C decreased protein yields insignificantly, whereas this tem- perature change re sulted in decreased y ields when the expression was under c ontrol o f the k promoters. This may be because pKE874 also directs low-level synthesis of a thermolabile version of the k cI repressor, which may repress transcription by E. coli RNAP at 30 °C. The optimized concentrations of T7 or E. coli RNAP used in the present work were found to be suitable for production of many different proteins. 4086 K. Ozawa et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Optimization of other conditions for in vitro protein synthesis Several other parameters were assessed individually to maximize protein yields. In particular, the optimal quan- tity of concentrated S30 e xtract in the r eaction mixture was determined for each new preparation, but results from several batches nevertheless gave similar results. In addition, the optimal concentrations of MgCl 2 and template DNA were found to vary with different batches of S30 extract. Concentration of the S30 extract by dialysis against a solution of PEG 8000 [2] reduced the volume of the in vitro reaction mixture, but had little effect on protein yields. The concentration of tRNA was found to affect the yields of proteins. For production of aspartyl-tRNA synthetase [16], for example, the optimal tRNA concentration was about 45 lgÆmL )1 , whereas tRNA concentrations of 87 and 175 lgÆmL )1 worked equally well for PpiB and hCypA. Proteins produced and stored in the reaction mixture appeared to b e stable with respect to proteolysis. After t wo months of storage at 4 °C, hCypA was not significantly degraded, as evaluated by NMR measurements. Cell-free synthesis of PpiB with i ncreasing concentra- tions of amino acids showed that almost n o protein was synthesized wh en e ach amino acid was present at 10 l M (Fig. 1 , lane 2). This result confirmed that t he extract is depleted in natural amino acids. Absence of free amino acids from t he cell extract is a prerequisite for efficient incorporation of labelled amino acids in to target proteins. Furthermore, the protein yields increased with increasing amino acid c oncentration ( Fig. 1, lanes 1 –8), indicating that the concentrations of amino acids limit protein synthesis at < 1 m M . After ultracentrifugation to remove ribosomes and ribosome-associated translation factors, the target proteins were f ound to be the most abundant protein in the reaction mixtures, provided the amino acids had been supplied a t high concentrations (Fig. 1). Remarkably, reduction of the concentration of amino acids to 30 l M lowered the yield only by abo ut 50% (Fig. 1, lanes 4 and 8). In vitro synthesis of h CypA selectively labelled with [ 15 N]amino acids The achievable y ields of p roteins would b e expected to depend on the concentrations of available amino-acylated tRNAs. As the loading efficiency of the amino-acyl tRNA synthetases depends on their K m values, amino acids processed by s ynthetases with low K m values are expected to be more readily available f or pr otein s ynthesis tha n others. To limit the e xpense of use o f labelled amino acids, their concentrations in the inner and outer chambers were adjusted according to their frequency in the primary structure of the protein and according to the K m values of their respective tRNA synthetases (Table 1). Our results confirmed that labelled amino acids from Groups I a nd II (Table 1) could b e used a t r educed concentrations (still several-fold above the respective K m values) witho ut signi- ficantly affecting protein yields. Figure 2 shows a compar- ison of yields obtained for hCypA with substantially reduced concentrations of Tyr, Thr and Asn, compared to Fig. 1. Cell-free sy nthesis of PpiB under control o f phage k promoters. Identical vo lumes of re action products were lo ade d in to lanes o f a 15% SDS/polyacrylamide gel, which were stained with Coomassie blue. Lanes1,3,5and7:reactionmixturesbeforethestartofin vitro synthesis of PpiB, with transcription by E. coli RNAP from tande m phage k promoters (0 h reactions). Lanes 2, 4, 6 and 8: corresponding mixtures after synthesis for 12 h at 37 °C. Each amino acid w as p resen t at a concentration of 10 l M (lanes 1 and 2), 30 l M (lanes 3 a nd 4), 300 l M (lanes 5 and 6) or 1 m M (lanes 7 and 8). Mobilities of molecular mass markers (kDa) were as indicated. Fig. 2. Cell-free synthesis of hCypA under control o f t he T7 pr omoter with minimized concentrations of labelled amino acids f rom the thre e different groups defined in Table 1. The samp les were centrifug ed for 4 h at 100 000 g before analysis by SDS/PAGE (15%). The gel was loaded with the soluble fractions (supernatants) of hCypA synthesized in vitro during 10 h a t 3 7 °C w ith d ifferen t con centrat ions o f 15 N-labelled a mino acids (all o ther amino acids were at 1 m M ), and stained with Coomassie blue. Lane 1 : 1 m M [ 15 N]Glu; lane 2: 50 l M [ 15 N]Tyr; lane 3: 150 l M [ 15 N]Thr; lane 4; 350 l M [ 15 N]Asn; lane 5 ; 1m M [ 15 N]His. These fractions were subjected to NMR measurements without further purification (Fig. 3). Mobilities of molecular mass markers (kDa) were as indicated. The [ 15 N]Glu-labelled hCYPA sample in lane 1 was produced with 200 m M ammonium acetate in the reaction buffer, whereas the samples in the other lanes were produced with 208 m M potassium glutam ate. Ó FEBS 2004 Cell-free synthesis of 15 N-labelled proteins (Eur. J. Biochem. 271) 4087 those obtained when all amino acids were at 1 m M .The similarity in protein production levels is corroborated by the similarity in cross-peak intensities observed in 15 N-HSQC NMR spectra recorded of the reaction mixtures (Fig. 3). The standard reaction mixture [2,15,16] contains a high concentration of potassium glutamate, which makes it unsuitable for selective labelling of Glu residues in target proteins. A buffer with 208 m M potassium D -glutamate performed equally well as one with L -glutamate, suggesting that glutamate served as a n o smolyte rather than p laying a more specific role. H owever, t his buffer w as still unsuitable for selective labelling with Glu, presumably because of the presence of glutamate racemase i n the S30 extract. The glutamate could not be substituted by 200 m M betaine, which h ad an inhibitory effect o n the synthesis of PpiB and hCypA. I n contrast, reaction mixtures with ammonium or potassium acetate instead of potassium glutamate w ere found to perform well. Maximal yields of P piB and hCypA were obtained with e ither acetate salt a t concentrations in the range of 200–230 m M (e.g. Figure 2 , lane 1). Further- more, this alternate medium suppressed amino- or amido- transferase activities which otherwise incorporated the a-amino-nitrogen of Asp into the a- and side-chain NH 2 groups of Asn (see below). 15 N-HSQC NMR spectra of selectively 15 N-labelled h CypA 15 N-HSQC spectra were recorded of hCypA samples produced in vitro with 19 different 15 N-labelled amino acids. Spectra w ith acceptable s ensitivity cou ld be r ecorded at 25 °C using the reaction mixtures at pH 7.5 (Fig. 3). Sample handling was kept to a minimum to explore the potential of this methodology for high-throughput protein analysis. Although spectra recorded before and after ultracentrifugation were not significantly different, all data presented in F ig. 3 were recorded after the ribosomes and other m acromole cular assemblies h ad been removed by ultracentrifugation to avoid the formation of a precipitate during data acquisition. The NMR chemical shifts of purified hCypA are k nown [27], allowing the identification of individual c ross-peaks f rom t he protein a nd detection of any additional cross-peaks due to metabolic by-products. The spectra recorded for hCypA produced with 15 N-labelled Asn, Gln, Ile, Leu, Phe and Tyr c ontained only cross-peaks from th e p rotein. Samples with 15 N-labelled A rg, A sp, His, Lys, Met, Thr and Val contained a few additional cross- peaks, due to limited metabolic conversion of the labelled amino acids. The additional peaks were, however, less intense than the average of the protein cross-peaks. Samples with 15 N-labelled Ala, Asp, Cys, Glu, Gly, Ser and Trp contained additional cross-peaks that were more intense than the average protein cross-peaks. In all cases, t he cross- peaks from metabolic by-products wer e removed easily by dialysis of the sample prior to NMR analysis (data not shown). F or proteins with a molecular mass similar to hCypA, dialysis can be achieved simply by transfer of the tubing containing the reaction mixture to a buffer suitable for N MR measurements. T he Sp ectra/Por 2 dialysis tubing (12–14 k Da cut-off) was found to retain a 9.5-kDa globular protein under these conditions. Comparison of spectra obtained with selectively 15 N-labelled PpiB and hCypA showed closely related sets of metabolite HSQC cross-peaks (data not shown). There was no evidence for transaminase activity except for the [ 15 N]Asp-labelled sample produced in the conven- tional way in the presence of potassium glutamate. Under those conditions, cross-peaks of the backbone and side- chain amides o f the [ 15 N]Asn-labelled sample w ere also observed in the NMR spectrum of[ 15 N]Asp-labelled hCypA (fourth panel of Fig. 3). The intensities of the undesired [ 15 N]Asn cross-peaks were about two thirds of those of the [ 15 N]Asp cross-peaks, indicating highly efficient transami- nation/transamidation. This suggests that the labelled amino group is not released in the form o f ammonia because i t would h ave been diluted by the presence of 27.5 m M ammonium present in the reaction buffer. The E. coli asparagine synthetases A and B (asnA and asnB gene products) s ynthesize A sn from Asp and may be r esponsible for this activity in the S30 extract. Remarkably, no evidence of transamination was observed i n the [ 15 N]Asn s ample. (The additional cross-peaks in Fig. 3 are due to the side- chain amides which were labelled in the [U- 15 N]Asn substrate). We further noted that the amido/aminotrans- ferase activity could be suppressed by replacing potassium glutamate in the buffer b y ammonium or potassium acetate (last p anel of Fig. 3). This new buffer did not affect the production levels of the proteins tested (hCypA, PpiB and ubiquitin) to a significant degree (e.g. see l ane 1 in Fig. 2). We also compared the buffers with 200 m M potassium acetate or 200 m M ammonium acetate for the synthesis of [ 15 N]Asp- and [ 15 N]Glu-hCypA with regard to the number and positions of HSQC cross-peaks from metabolites. No significant differences were observed (data not shown). Quite generally, t he cell-free expression system allows the use of h igh c oncentrations of unlabelled amino acids i n t he reaction mixtures to dilute the effects of incorporation of amino acids that are 15 N-labelled by transamination. In this situation, the main effect of transamination is a reduction of the degre e of 15 N-labelling in the amino a cid of interest. In thecaseof[ 15 N]Gly, much of the label appeared to have been trapped in an a mide group of a low molecular mass metabolite (Fig. 3). Fig. 3. 15 N-HSQC spe ctra of hCypA selectively labelled with 15 N-amino acids. The spectra were recorded at 25 °C and pH 7.5 using the in vitr o reaction mixture after centrifugation (100 000 g, 4 h) and addition of 10% D 2 O. The assignments of the backbone a mide cross- peaks are indicated by the one-letter amino acid symbols and the sequence numbers. Sq uares i dentify the c ross peaks which could b e assigned acc ording to the previously publ ished assignment [27]. Circles identify the cross-peaks from metabolites. Dotted squares mark the p osit ions of cross-peaks w hic h were assigned at pH 6.5 [27] but are absent from the present spectra or observable only at lower plot levels. The spectrum recorded with [ l5 N]Asn also contains the cross-peaks from t he side-chain amide gr oups (backbone and side-chain NH groups were labelled in the amino acid used in the reaction). Th e cross-peak from the side-chain N H of W121 i s labelled W121e. Question marks in the spectra of [ 15 N]Met and [ 15 N]Val hCypA identify tentative n ew assignments of cross-peaks which had n ot been assigned previously [27]. 4088 K. Ozawa et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Ó FEBS 2004 Cell-free synthesis of 15 N-labelled proteins (Eur. J. Biochem. 271) 4089 Fig. 3. (Continued). 4090 K. Ozawa et al.(Eur. J. Biochem. 271) Ó FEBS 2004 The cell-free expression system lends itself to t he addition of specific enzyme inhibitors, such as the alanine racemase inhibitor, b-chloro- L -alanine [28]. When we t ested the effect of adding b-chloro- L -alanine (0.5 m M ) to the reaction mixture for the synthesis of [ 15 N]Ala labelled PpiB, however, only a single cross-peak belonging to a minor metabolite was removed from the set of metabolite signals (data not shown). The metabolic enzymes and pathways in E. coli are not sufficiently well understood to suppress all side reactions in this way. Single additional peaks present in the 15 N-HSQC spec- trum of [ 15 N]Met and [ 15 N]Val labelled hCypA were still present after dialysis and were attributed to the amide protons of Met1 and Val2. These h ad not been reported in the original assignment of hCypA [27], in agreement with the frequent observation that the H N resonances of amino- terminal residues are broadened beyond detection by fast base-catalyzed exchange with the water. In t he case of our cell-free expression system, the amino-terminal Met of PpiB has been observed (by proteolysis and mass spectrometry) to remain predominantly N-formylated due to saturation of the activity of p eptide deformylase ( D. Mouradov and T. Huber, unpublished data). I n the present case of hCypA, retention of the N-formyl group enables the ob servation o f the cross-peaks from the amino-terminal amide protons. Interestingly, ESI mass spectrometric analysis of hCypA produced in vivo in E. coli also showed N-terminal hetero- geneity. Although  70% of the protein s tarted with a deformylated Met (18 012 Da),  20% had N-terminal N-formyl-Met (18 040 Da) and  10% had l ost the N-terminal Met (17 881 Da ). In our experiments, weak cross-peaks with broad line shapes were difficult to detect. Hence not all signals assigned previously [27] could be observed. As the original assign- ments h ad been reported f or pH 6.5, we measured the 15 N-HSQCspectraafterdialysisatthispH.ThelowerpH value enhanced some of the signals as expected. For example, the cross-peak of Asp9 was observed at pH 6.5 (data not shown), whereas it was missing from the spectrum recorded at pH 7.5 (Fig. 3). In contrast to the previously reported assignment [27], there was no evidence for more than a s ingle conformation of His70, neither at pH 7.5 nor at pH 6.5. Discussion In this study, several aspects of a cell-free protein synthesis system b ased on an E. coli cell extract were investigated and optimized. O ne po int is the observation that T7 RNA polymerase performs as well as the E. coli holoenzyme in the in vitro coupled transcription/transla- tion system. As many modern plasmid vectors used for protein overproduction are based on transcription from the T7 promoter, it is convenient if the same vectors can be used in the c ell-free system. In the case of hCypA, good protein yields were obtained with transcription by T7 RNAP from the T7 promoter, whereas no p roduction could be detected in vitro with a phage k promoter and transcription b y the E. coli RNAP holoenzyme. However, no difference between the two systems was observed for the closely related protein PpiB. This phenomenon may be explained in different ways. Possibly, hCypA has less favourable folding characteristics than PpiB. Alternat- ively, the relative c omplexity of t he E. coli RNAP holoenzyme may offer more targets fo r inhibitory protein–protein interactions. Whereas t he present study required s ignificant NMR measurement time to record each 15 N-HSQC spectrum, this is no longer prohibitive with the increased sensitivity available on high-field NMR spectrometers equipped with cryogenic probeheads [16]. We thus envisage that parallel production of a large number of selectively l abelled p rotein samples followed by the recording of 15 N- or 13 C-HSQC spectra will provide a practical approach to support resonance assignments, p rovided that sample handling c an be kept to a minimum. The present study shows that th is latter condition is easily fulfilled. For most amino acids, purification of the prod uced 15 N-labelled p rote in is not required for identification of the HSQC cross-peaks from the protein. In the few cases where cross-peaks from metabolites and protein could overlap, a simple dialysis step is sufficient to r emove the metabolites. The easy removal of the i nterfering signals from metabolites is a clear advantage of the cell-free expression system over in-cell NMR analyses [29,30]. Interestingly, only low-mass metabolites appear to be produced also when [U- 15 N]protein is s ynthesized in vivo using ammonium chloride [31]. Most importantly, the transf er of the 15 N-label t o other amino acids is insignificant for 1 8 of the amino acids and can b e suppressed for [ 15 N]Asp b y use of a modified medium in which glutamate is replaced by acetate. Notably, these r esults were achieved without preparation of extracts from auxotrophic E. coli strains. Replacement of glutamate by acetate has recently also been described by Klammt et al.[7]. Cell-free expression kits for large-scale protein produc- tion have become commercially available, making cell-free expression a r eadily accessible technology [7,11,32]. Our strategy extends the applicability of t he system with regard to protein analysis by NMR spectroscopy. The catalogue of 15 N-HSQC spectra in Fig. 3 provides the basis for the straightforward identification of metabolite cross-peaks by visual inspection, as their generation i s insensitive to the identity of the target protein. Ultimately, the formation of metabolites could be avoided by the use of a completely reconstituted cell-free system comprising only the minimum set of enzymes necessary for translation [33]. Currently achievable yields, however, do not justify the effort associated with the purification of the l arge number of enzymes required. If all metabolites generated by the E. coli S30 extract could be identified, it might b e possible t o suppress t heir production by the use of appropriate enzyme inhibitors. This would provide the benefit of less isotopic dilution and thereby improved labelling efficiency and enhanced sensitivity of the NMR analysis. 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Supplementary material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4346/EJB4346sm.htm Details of the construction of the synthetic s-CYPA gene, purification of cyclophilin A and expression and purification of T7 RNA polymerase a re given and reference to the following supplementary figures is made: Fig. S 1. Construction of the synthetic CYPA gene (s-CYPA). The gene was constructed following manipula- tion of the nucleotide sequence t o increase its identity with the E. coli ppiB gene, using a combination of ligation and recursive overlap extension of complementary synthetic oligonucleotides as described above. Oligonucleotides used for the construction of the s-CYPA are i dentified by arrows above the complete gene sequence. T he Nd eI, Apa I, FokI, XhoI, EcoRI and NcoI restriction endonuclease sites are boxed. The start codon (ATG) is within t he NdeIsiteandthe stop codon (TAA) is i dentified by a black box in the linker. Fig. S 2. Plasmid pKO1166. T his plasmid, which directs overproduction of T7 RNA polymerase, was constructed by insertion of a DNA fragment bearing T7 gene 1 under control of the bacteriophage k p L promoter into vector pMA200U [5]. Ó FEBS 2004 Cell-free synthesis of 15 N-labelled proteins (Eur. J. Biochem. 271) 4093 . Optimization of an Escherichia coli system for cell-free synthesis of selectively 15 N-labelled proteins for rapid analysis by NMR spectroscopy Kiyoshi. to the preparation of 15 N-labelled samples of hCypA and analysis by NMR spectroscopy, the performance of the cell-free expression system was explored