A novel cell-free protein synthesis system

Journal of Biotechnology 110 (2004) 257–263

A novel cell-free protein synthesis system Kalavathy Sitaraman a , Dominic Esposito a , George Klarmann b , Stuart F. Le Grice b , James L. Hartley a , Deb K. Chatterjee a,∗ a

SAIC-National Cancer Institute at Frederick, 1050 Boyles Street, Building 327, Frederick, MD 21702-1201, USA b HIV Drug Resistance Program, 1050 Boyles Street, Building 327, Frederick, MD 21702-1201, USA Received 17 September 2003; received in revised form 19 February 2004; accepted 27 February 2004

Abstract An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications. © 2004 Elsevier B.V. All rights reserved. Keywords: Cell-free protein synthesis; 3-Phosphoglycerate; PEP; Gam protein; RecBCD; T7 RNA polymerase

1. Introduction A key to a clear understanding of the role of genes in an organism is to understand the functions of all its proteins (biochemical activities and protein–protein interactions) at the molecular level. Acquiring this knowledge will depend, in part, on the rapid expression and purification of proteins on a large scale and with high throughput. Accomplishing such rapidity will be far more difficult than the sequencing of genomes because the behavior of proteins is no∗ Corresponding author. Tel.: +1-301-846-6893; fax: +1-301-846-6631. E-mail address: [email protected] (D.K. Chatterjee).

toriously variable. Many molecular tools enable in vivo protein expression in host organisms such as Escherichia coli and other eukaryotic cells. However, the labor required to express many genes is considerable and the reasons for failure are numerous, including insolubility, toxicity to the host, and instability (Service, 2002). These considerations have led to attempts to bypass the intact host organism and to use instead cell extracts that contain the essential transcription–translation factors and to simply add the DNA template to the extract and let expression proceed in vitro. In vitro protein synthesis has advantages in producing only the desired protein without producing any undesired proteins that are required for cell maintenance

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.02.014

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in vivo. Because it is essentially free from cellular regulation of gene expression, in vitro protein synthesis has advantages in the production of cytotoxic, unstable, or insoluble proteins. In addition, various kinds of unnatural amino acids can be efficiently incorporated into proteins for specific purposes (Noren et al., 1989). Despite all its promising aspects, the in vitro protein synthesis system has not been widely accepted as a practical alternative, in part due to the short reaction period which results in poor yields of protein. A steady supply of ATP is one of the keys for efficient synthesis of proteins in cell-free systems. The most popular secondary energy source to generate ATP in E. coli cell-free system has been phosphoenol pyruvate (PEP) (Zubay, 1973). However, acetyl phosphate (Ryabova et al., 1995) and creatine phosphate (Kigawa et al., 1999) have also been used. Unfortunately, all of these secondary energy sources are unstable in E. coli extracts due to unwanted enzymatic degradation (Kim and Swartz, 1999, 2001; Kim and Choi, 2000). Other reasons may be loss of NTPs (Kawarasaki et al., 1994; Nakano et al., 1996) and mRNA instability (Kitaoka et al., 1996; Ueda et al., 1991). A number of subsequent improvements have been attempted (Kim et al., 1996; Patnaik and Swartz, 1998; Kim and Swartz, 2001) to improve the cell-free protein synthesis system. However, these systems still do not produce sufficient quantities of protein for extensive analysis of proteins of interest. In addition, the problem of DNA template stability is especially evident when linear substrates such as PCR derived products are used in cell-free extracts for generating proteins. The linear DNA fragments are susceptible to rapid degradation by intracellular exonucleases of E. coli, particularly RecBCD (Pratt et al., 1981). In most cases, a super-coiled plasmid DNA containing the gene of interest is used in cell-free systems because plasmid DNAs are more stable (Kudlicki et al., 1992). Mutant recBCD strains devoid of the exonuclease have been made. These mutant strains do not as rapidly degrade linear DNA; however, such mutant strains grow extremely poorly and therefore do not produce satisfactory results (Yu et al., 2000). In this report, we demonstrate the usefulness of 3-phosphoglycerate (3-PGA) as a secondary energy source in a cell-free protein synthesis system using

E. coli cell extract. Protein synthesis continues for a longer time period with 3-PGA compared to PEP, thereby resulting in more protein. In addition, we show that addition of bacteriophage ␭ Gam protein, an inhibitor of RecBCD, stabilizes linear PCR DNA template in E. coli extracts and produces comparable amounts of protein as one would expect from plasmid templates. Combination of these two improvements should allow synthesis of considerable amount of proteins suitable for high-throughput analysis as well as structural investigation in the post-genomic era.

2. Materials and methods 2.1. Reagents Phosphoenol pyruvate, 3-phosphoglycerate and E. coli t-RNA mixture and other reagents were purchased form Sigma (St. Louis, MO). Green fluorescent protein (GFP) was purchased from Roche Biologicals. T7 RNA polymerase was either purified from a clone using affinity chromatography or obtained from commercial sources (Invitrogen or US Biochemical). Plasmid pUCT7-CAT and pUCT7-GFP (unpublished) were used as control templates for CAT and GFP synthesis. 2.2. Reaction protocols Extracts were prepared from E. coli BL21 and BL21-Rosetta (Novagen, Madison, USA) according to Zubay (1973), except that the cells were disrupted by a high-pressure homogenizer (Avestin, Canada). The standard reaction mixture consisted of the following components: 57 mM Hepes-KOH, pH 7.6, 230 mM k-glutamate, 80 mM ammonium acetate, 2 mM each of ATP and GTP, 0.85 mM each of CTP and UTP, 30–40 mM 3-phosphoglycerate or PEP (when used for comparison), 1.7 mM DTT, 12 mM Mg-acetate, 0.17 mg/ml E. coli t-RNA mixture, 34 ␮g/ml folinic acid, 30–50 ␮g/ml T7 RNA polymerase, 1 mM of each amino acids, 10–20 mg/ml extract, 2% polyethylene glycol, 0.65 mM cAMP, and various amounts of DNA. The reaction was carried out at either 30 or 37 ◦ C for 2–3 h. Following reaction, 0.5–1 ul of the reaction product was analyzed by 4–20%

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polyacrylamide gel electrophoresis. For radioactive amino acid (S35 -methionine) incorporation, RNaseA (5 ug) was added and reactions were incubated at 37 ◦ C for 15 min. Reactions (5 ul) were spotted on GFC filters, washed once in 10% trichloroacetic acid, twice in 5% trichloroacetic acid and finally, once in methanol. Filters were dried, placed in 4 ml Scintisafe-F, and counted for 1 min. The amount (pmoles) of incorporated methionine for each sample is a measure of protein synthesis. 2.3. Cloning and purification of Gam protein A short form of bacteriophage lambda gam (gamS) was amplified using the primers 5 -GGGAGGCCATGGGAAACGCTTATTACATTCAGGATCGTCTTG-3 and 5 -GGGAGGAGATCTTTATACCTCTGAATCAATATCAACC-3 . The amplified product was cleaved with NcoI and BglII and cloned into a pBAD-His vector (Invitrogen) cut with the same restriction enzymes. The final clone pLDE136 was introduced into DH10B (Invitrogen). An overnight culture was diluted 50-fold in LB-Amp in a 50 ml flask, and grown to an OD of 0.6 at 37 ◦ C. Arabinose was added to a final concentration of 0.2%, and the cells were grown for an additional 3 h before harvesting. The pellet was frozen, thawed, resuspended in 500 ␮l extract buffer (50 mM Tris–Cl, pH 8.0, 0.1 mM EDTA, 10% glycerol, 500 mM NaCl), sonicated (Ultrasonic) twice for 30 s using a 0.3175 cm probe at 50% power, and clarified by centrifugation. The sample was diluted to 100 mM NaCl prior to chromatography. A 1 ml Hitrap-MonoQ column (Pharmacia) was equilibrated with 10 ml of buffer B (50 mM Tris–Cl, pH 8.0, 0.1 mM EDTA, 10% glycerol, 1 M NaCl, 1 mM DTT) and then with 10 ml of buffer A (50 mM Tris–Cl, pH 8.0, 0.1 mM EDTA, 10% glycerol and 1 mM DTT), before being washed extensively with 10% buffer B. The extract was loaded at 10% buffer B at a flow rate of 0.5 ml/min, washed with 10% buffer B at 0.5 ml/min for 15 CV, and eluted with a 20 CV gradient of 10–70% buffer B at 0.25 ml/min. Fractions were collected every 0.5 ml. GamS eluted in three main fractions at a salt concentration of approximately 400 mM. These fractions were analyzed on SDS-PAGE, and pooled into a single Mono Q pool. By Coomassie staining, this pool was estimated to be 70–80% pure.

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2.4. Activity assay for GamS-mediated RecBCD inhibition We have developed a radioactive assay to measure GamS activity. Briefly, a uniformly 32 P-labeled double-stranded DNA (50 fmol) was used as the substrate for RecBCD (Plasmid-safe ExoV, Epicentre). Gam S (0–200 ng) was preincubated with 1 unit RecBCD and 10 mM ATP for 10 min at 37 ◦ C, and then added to the DNA in a total volume of 100 ␮l. After different time of incubation reactions were stopped by the addition of 100 ␮l of binding buffer (6 M guanidine and 100 mM MES buffer). Reaction mixtures (150 ␮l) were spotted onto GFC filters, washed and counts remained bound in the filters were determined. The Mono Q pool (above) was assayed to determine the amount of Gam activity present. It had ∼25–30 units/ul, and was 194 ng/ul in protein concentration, giving a specific activity value between 160 and 200 units/mg of GamS (assuming 70–80% purity).

3. Results and discussions 3.1. 3-Phosphoglycerate as an energy source The biochemical energy source in an in vitro protein synthesis system is derived from the hydrolysis of triphosphates, the concentration of which is maintained by an energy regeneration system. The conventional secondary energy sources such as PEP, acetyl phosphate or creatine phosphate are unstable due to their unproductive depletion by phosphatases present in the E. coli extract (Kim and Swartz, 1999, 2001). This results in the early cessation of protein synthesis. The typical amount of secondary energy source used in the system was 30–40 mM. This concentration perhaps was much higher than the theoretical Km for the phosphatases and thus, phosphatases could use them as their substrates. We reasoned that if PEP could be synthesized in situ using a glycolytic intermediate, at any given time the concentration of PEP would be so low that phosphatases could not use it as a substrate. The net result would be sustained synthesis of ATP and the reaction would continue for a longer time yielding more protein. We have used 3-phosphoglycerate to test our hypothesis and phosphoenol pyruvate, a commonly used

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tion continues for more than 2 h when 3-PGA was used as a secondary energy source. In comparison, the reaction rate slowed down considerably between 30 and 45 min when PEP was used under identical conditions (Fig. 2). Early cessation of protein synthesis and thus, the lower yield using PEP can be explained by unproductive loss of PEP due to non-specific phosphatase attack as observed by Kim and Swartz (1999). Other secondary energy sources, such as acetyl phosphate and creatine phosphate were also shown to be degraded by non-specific phosphatases present in the E. coli extracts (Kim and Swartz, 1999, 2001). On the other hand, 3-PGA appears to be more stable in E. coli cell extract and the yield was estimated to be more than a milligram of protein per milliliter of reaction

3-phosphoglycerate phosphoglycerate mutase

2-phosphoglycerate enolase

Phosphoenolpyruvate ADP pyruvate kinase

ATP Pyruvate Fig. 1. Biochemical pathway used to generate ATP using 3-phosphoglycerate (3-PGA) as an energy source.

secondary energy source was used for comparison. 3-PGA is converted to PEP by two sequential enzymatic reactions catalyzed by phosphoglycerate mutase and enolase, respectively (Fig. 1). In a real time study of GFP expression, we show (Fig. 2) that the reac-

30,000

3-PGA

Fluorescence Units

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time (min) Fig. 2. Real-time monitoring of green fluorescent protein measure by fluorescence emission in the presence of 3-phosphoglycerate (3-PGA) or the conventional phosphoenolpyruvate (PEP) as the energy source. Expression levels were measured at 5 min intervals in real time. Reactions were done in triplicate for each substrate.

Fig. 3. Polyacrylamide gel analysis of protein synthesized after 2 h in the real time expression study (Fig. 2) using 3-PGA or PEP as an energy source. One microliter of the reaction product was loaded onto the gel. A reaction without any DNA template was used as a reference (R).

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3.2. Gam protects PCR DNA templates in cell extracts For high-throughput synthesis of proteins, use of PCR products has several advantages, because both cloning and purification of plasmid templates can be completely eliminated. This can result in rapid analysis of coding sequences. However, the problem of DNA template stability is evident when linear substrates, such as PCR derived products, are used in cell-free extracts. The linear DNA fragments are susceptible to rapid degradation by intracellular exonucleases of E. coli, particularly RecBCD (Pratt et al., 1981; Lorenz and Wackernagel, 1994) and possibly by other nucleases. This problem leads to very low levels of protein production. Analysis of cell-free extracts from an E. coli BL21-Rosetta strain using a radioactive exonuclease assay showed that there are significant levels of RecBCD-like exonuclease activity in the extracts (approximately 0.4–0.8 units/␮l extract, unpublished). Bacteriophage ␭ produces a protein called Gam, which is known to inhibit the RecBCD nuclease activity (Yu et al., 2000). Linear DNA (PCR fragment) can be completely protected against purified RecBCD in the presence of purified GamS, the short form of Gam protein (Murphy, 1991), for up to 4 h of incubation (Fig. 4). However, in experiments with crude

exo-resistant counts (x10 3 )

which is almost double the amount compared to PEP as energy source (Fig. 3). To improve protein synthesis, Kim and Swartz (1999, 2001) used glucose 6-phosphate and pyruvate, the first and the last intermediates of the glycolytic pathways, as secondary energy sources and compared with most commonly used energy source PEP, another glycolytic intermediate. It was thought that glucose 6-phosphate would produce more ATP during its oxidation to pyruvate (three molecules of ATP per molecule of glucose 6-phosphate compared to one per molecule PEP) and thus, will produce more protein (theoretically almost three times). However, only about 30% improvement over PEP (with two co-factors, NAD and CoA) was observed. The authors reasoned that marginal improvement may be due to degradation of glucose 6-phosphate by phosphatases in E. coli extract. In comparison, 3-PGA without any additional co-factors synthesized almost twice (100% improvement) the amount of protein compared to PEP. Since the reaction condition used in our studies is similar to Kim and Swartz except that there were no co-factors, it can be argued that 3-PGA is more stable in E. coli extract than glucose 6-phosphate or its intermediates prior to 3-PGA. It is also possible that as PEP was synthesized in situ from 3-PGA, it was used mainly for ATP production before phosphatase attack. It has been reported that pyruvate alone can produce protein, but only about 20% that of PEP derived protein (Kim and Swartz, 2001), while in the presence of two co-factors (NAD and CoA), this number increases to 70%. Taken altogether, 3-PGA should be a better alternative to glucose 6-phosphate, PEP or pyruvate for secondary energy source in the cell-free protein synthesis system because it is more stable, produces more protein and does not require any additional co-factors. In addition to the improvement in the energyregenerating system, we have also used cell extracts from E. coli BL21-Rosetta (Section 2). This strain overproduces t-RNAs for five rare codons (AGG/AGA, CCC, AUA, CUA and CCC) found mostly in eukaryotic genes and thus, might be useful for producing proteins with rare codons. In fact, in several cases, the extract from this strain produced more proteins containing rare codons (data not shown) compared to the extract made from E. coli BL21 or A19, the most commonly used strains.

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100 80 60 40

No GamS 10 ng GamS

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time (min) Fig. 4. Stabilization of PCR product DNA template by GamS in the presence of purified RecBCD protein. GamS (0, 10 or 200 ng) were incubated with 1 unit of RecBCD and the reaction was processed as described in Section 2. The y-axis shows the number of exonuclease resistant counts retained on the filter at each time point.

exo-resistant counts

100% 30 ng 2 ng 0 ng

80% 60% 40% 20% 0% 0

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time (min) Fig. 5. Protection of linear DNA template in E. coli extracts in the presence of GamS. Radiolabeled linear PCR product along with the indicated amount of GamS protein (0, 2 or 30 ng) was added to a standard reaction, and samples were taken out at the indicated times. The y-axis indicates the relative percentage of exonuclease resistant counts retained on the filter. 100% represents the control value at time zero of approximately 100,000 counts.

extracts, lengthening the incubation time leads to the degradation of PCR template, independent of the levels of GamS added. After 30 min, 80% of the template remained; after 2 h of incubation, only 30% of the template remained (Fig. 5). In the absence of GamS, the template is completely degraded within 30 min. It is therefore likely that other E. coli nucleases that are not inhibited by Gam, (such as ExoIII, ExoVIII, EndoIV and double stranded DNA specific nucleases) are also acting on the linear PCR DNA template. Therefore, mutations or inhibition of any or all of these genes may be useful to protect linear templates for longer times. We are in the process of making the mutations of these unwanted genes to further improve template stability. 3.3. Gam increases protein production from PCR DNA templates Cell-free in vitro transcription–translation reactions (50 ␮l) were prepared as described in the Section 2 using PCR DNA templates (50 ng). Cell-free protein synthesis was followed in the presence (5 units or 200 ng protein) or absence of GamS protein (Fig. 6). In the absence of Gam protein, BL21-Rosetta cell extract yields from a PCR product DNA template is decreased (2–3-fold) as compared to the yield from a supercoiled DNA plasmid template. Addition of GamS protein to the reaction has very little effect on synthesis from the supercoiled template. The effect of GamS on protein

S-met incorporated (pmol)

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plasmid

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Fig. 6. Synthesis of CAT protein from templates of different topologies in the presence or absence of GamS. Reactions were carried out using 50 ng of either supercoiled plasmid or linear PCR product in the absence of GamS, or with 200 ng of GamS for 2 h at 37 ◦ C.

production from the PCR product DNA template was dramatic, raising the yield to a level comparable to that observed with supercoiled DNA template. In summary, we have developed a highly efficient cell-free protein synthesis system using a novel glycolytic intermediate, 3-phosphoglycerate, capable of yielding twice as much protein compared to the phosphoenolpyruvate system in a simple batch reaction. It is worth mentioning that the cost of 3-PGA is only a fraction of the cost of conventional phosphoenol pyruvate, while the yield of protein is doubled with 3-PGA as energy source. In addition, we have shown that in the presence of GamS, an inhibitor of E. coli RecBCD, protein synthesis can be achieved by simple addition of PCR product to the cell-free extract. In addition, cell extracts from BL21-Rosetta containing overexpressed rare t-RNAs might be useful for producing eukaryotic proteins containing rare codons. Combination of these improvements should be extremely useful for rapid expression and purification of proteins on a large scale in a high-throughput manner for functional and structural analysis.

Acknowledgements This project has been funded in whole or in part with Federal funds from the National Cancer

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Institute, National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. A part of this work was initiated at Invitrogen/Life Technologies, Inc. The authors thank Mary Longo and Karen Van Orden for technical support.

References Kawarasaki, Y., Nakano, H., Yamane, T., 1994. Prolonged cell-free protein synthesis in a batch system using wheat germ extract. BioSci. Biotech. Biochem. 58, 1911–1913. Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., Yokoyama, S., 1999. Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19. Kim, D., Swartz, J., 1999. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotech. Bioeng. 66, 180–188. Kim, D., Kigawa, T., Choi, C., Yokoyama, S., 1996. A highly efficient cell-free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239, 881–886. Kim, R., Choi, C., 2000. Expression-independent consumption of substrates in cell-free expression system from Escherichia coli. J. Biotech. 84, 27–32. Kim, D., Swartz, J., 2001. Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotech. Bioeng. 74, 309–316. Kitaoka, Y., Nishimura, N., Niwano, M., 1996. Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system. J. Biotech. 48, 1–8.

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Kudlicki, W., Kramer, G., Hardesty, B., 1992. High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system. Anal. Biochem. 206, 389–393. Lorenz, M., Wackernagel, W., 1994. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563–602. Murphy, K., 1991. Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J. Bacteriol. 173, 5808–5821. Nakano, H., Tanaka, T., Kawarasaki, Y., Yamane, T., 1996. An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. Biosci. Biotechnol. Biochem. 58, 631–634. Noren, C., Anthony-Cahill, S., Griffith, M., Schultz, P., 1989. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188. Patnaik, R., Swartz, J., 1998. E. coli-based in vitro transcription/translation: in vivo-specific synthesis rates and high yields in a batch system. Biotechniques 24, 862–868. Pratt, J., Boulnois, G., Darby, V., Orr, E., Wahle, E., Holland, I., 1981. Identification of gene products programmed by restriction endonucleases DNA fragments using an E. coli in vitro system. Nucleic Acids Res. 9, 4459–4474. Ryabova, L., Vinokurov, L., Shekhovtsova, E., Alakhov, Y., Spirin, A., 1995. Acetyl phosphate as an energy source for bacterial cell-free translation systems. Anal. Biochem. 226, 184–186. Service, R.F., 2002. Structural genomics. Tapping DNA for structures produces a trickle. Science 298, 948–950. Ueda, T., Tohda, H., Chikazumi, N., Eckstein, F., Watanabe, K., 1991. Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. Nucleic Acids Res. 19, 547–552. Yu, D., Ellis, H., Lee, E., Jenkins, A., Copeland, N., Court, D., 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. 97, 5978–5983. Zubay, G., 1973. In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–287.