Cell-Free Protein Expression and Functional Assay in Nanowell

The RTS 100 Escherichia coli HY kit was obtained from Roche Diagnostics GmbH ... Following expression, the mix was diluted 1:10 in PBS and the inhibit...
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Anal. Chem. 2004, 76, 1844-1849

Cell-Free Protein Expression and Functional Assay in Nanowell Chip Format Philipp Angenendt,*,†,‡ Lajos Nyarsik,† Witold Szaflarski,† Jo 1 rn Glo 1 kler,† Knud H. Nierhaus,† Hans Lehrach,† Dolores J. Cahill,‡,§,| and Angelika Lueking*,†,‡

Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany, Ruhr-University Bochum, Medical Proteome Center, Universita¨tsstrasse 150, 44780 Bochum, Germany, PROTAGEN AG, Emil-Figge-Strasse 76A, 44227 Dortmund, Germany, and The Centre for Human Proteomics, Royal College of Surgeons in Ireland, Dublin 2, Ireland

The expression and characterization of large protein libraries requires high-throughput tools for rapid and costeffective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology. In the past few years, major progress in miniaturization and parallelization of screening assays has been achieved. The combination of miniaturization with automation, sensitive signal detection, various plate formats, automated compound delivery, and data management enables efficient high-throughput analysis in clinical diagnostics, genomics, proteomics, and pharmaceutical screening. As one of the first products of this miniaturization trend, the microarray technology emerged in the 1990s.1-3 Originally developed for the profiling of gene expression patterns, the technology has become a valuable research tool and is today also commonly applied for sequencing4 and SNP analysis.5 However, the need for high-throughput technologies for the analysis of the * To whom correspondence should be addressed. Telephone: +49 (0) 30 8413-1648 or -1631. Fax: +49 (0) 30 8413 1128. E-mail: [email protected]; E-mail: [email protected]. † Max-Planck-Institute for Molecular Genetics. ‡ Ruhr-University Bochum. § PROTAGEN AG. | Royal College of Surgeons in Ireland. (1) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 46770. (2) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-6. (3) Brown, P. O.; Botstein, D. Nat. Genet. 1999, 21, 33-7. (4) Diamandis, E. P. Clin. Chem. 2000, 46, 1523-5.

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proteome led to the adaptation of DNA microarray principles to protein science and resulted in the development of protein, antibody, and cell microarrays.6-10 Microarrays consist of an ordered arrangement of biomolecules, which are immobilized at high density onto a chemically modified surface.11 Although a multitude of different surface chemistries have been developed,12,13 the immobilization is still a major problem, since many proteins are prone to lose their function and activity by surface attachment.14-16 Hence, many screening assays and functional and metabolism studies are performed in small-volume reaction vessels today, avoiding immobilization of the target protein and, consequently, circumventing potential loss of activity. Currently, 96- and 384-well microtiter plates are the standard format for high-throughput procedures using ∼100-µL sample volume.17,18 However, assuming a typical pharmaceutical screening run with 100 000 compounds, this format would necessitate the consumption of at least 10 L of target solution. Additionally, the minute amounts of compounds available are needed for many different assays. The same holds true for the discovery of enzymes with novel properties. Biotechnology is in constant need to discover new enzymes, e.g., to produce drugs in a stereoselective way, since some contaminating enan(5) Cutler, D. J.; Zwick, M. E.; Carrasquillo, M. M.; Yohn, C. T.; Tobin, K. P.; Kashuk, C.; Mathews, D. J.; Shah, N. A.; Eichler, E. E.; Warrington, J. A.; Chakravarti, A. Genome Res. 2001, 11, 1913-25. (6) Lueking, A.; Horn, M.; Eickhoff, H.; Bu ¨ ssow, K.; Lehrach, H.; Walter, G. Anal. Biochem. 1999, 270, 103-11. (7) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2, RESEARCH0004. (8) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Anal. Biochem. 2003, 312, 113-24. (9) Schwenk, J. M.; Stoll, D.; Templin, M. F.; Joos, T. O. Biotechniques 2002, (Suppl), 54-61. (10) Angenendt, P.; Glokler, J.; Konthur, Z.; Lehrach, H.; Cahill, D. J. Anal. Chem. 2003, 75, 4368-72. (11) Glokler, J.; Angenendt, P. J Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003, 797, 229-40. (12) Angenendt, P.; Glo ¨kler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253-60. (13) Angenendt, P.; Glokler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr., A 2003, 1009, 97-104. (14) MacBeath, G. Nat. Genet. 2002, 32, 526-32. (15) Cahill, D. J. J. Immunol. Methods 2001, 250, 81-91. (16) Angenendt, P.; Glo ¨kler, J. In Methods in Molecular Biology; Fung, E., Ed.; The Humana Press: Totowa, NJ, 2004. (17) Garyantes, T. K. Drug Discovery Today 2002, 7, 489-90. (18) Masimirembwa, C. M.; Thompson, R.; Andersson, T. B. Comb. Chem. High Throughput Screen 2001, 4, 245-63. 10.1021/ac035114i CCC: $27.50

© 2004 American Chemical Society Published on Web 02/25/2004

Figure 1. Left: Photograph of the nanowell plate comprising 12 × 8 wells with a well volume of 1.5 µL. Right: Enlarged photograph of the nanowells with a spacing of 2.25 mm between each well.

tiomeres may even have adverse effects.19 Large libraries of either mutant variants of known enzymes or enzymes of metagenomic origin are to be screened.20,21 As a consequence, further miniaturization of high-throughput devices is necessary to save solutions and precious compounds. Therefore, assays in volumes ranging from 1 to 5 µL are currently under investigation using plates with 1536 wells or more.22 Those microfabricated carrier plates have been shown to be useful as containers for electrokinetic injections of DNA samples, MALDITOF-MS analysis of oligonucleotides or proteins, and enzymatic digestion of proteins.23 Combined with highly sensitive fluorescence resonance energy transfer-based detection, chymotrypsin cleavage of a fluorophore-labeled peptide was shown in a 3456well assay format. This format was applied to a cell-based receptor assay, showing the dose-dependent interaction of the muscarinic M1 receptor with its agonist carbachol.24 Here, we have developed a new approach combining nanowell technology with in vitro protein synthesis based on a cell-free translation system.25 Such cell-free systems offer several advantages superior to traditional cell-based expression systems, such as expression of toxic or insoluble proteins.26 Moreover, initial attempts with the production of poorly expressed proteins suggest new applications of cell-free systems.27 We demonstrate the adaptation of one of them (Rapid Translation System 100, RTS 100) to the nanoliter-scale format and subject those in vitro synthesized proteins to enzymatic assays. Furthermore, we show the usefulness of such a format with an inhibition assay to (19) De Camp, W. H. Chirality 1989, 1, 2-6. (20) Koga, Y.; Kato, K.; Nakano, H.; Yamane, T. J. Mol. Biol. 2003, 331, 58592. (21) Lorenz, P.; Liebeton, K.; Niehaus, F.; Eck, J. Curr. Opin. Biotechnol. 2002, 13, 572-7. (22) Wo ¨lcke, J.; Ullmann, D. Drug Discovery Today 2001, 6, 637-46. (23) Litburn, E.; Emmer, A.; Roeraade, J. Electrophoresis 2000, 21, 91-9. (24) Mere, L.; Bennett, T.; Coassin, P.; England, P.; Hamman, B.; Rink, T.; Zimmerman, S.; Negulescu, P. Drug Discovery Today 1999, 4, 363-9. (25) Spirin, A. S.; Baranov, V. I.; Ryabova, L. A.; Ovodov, S. Y.; Alakhov, Y. B. Science 1988, 242, 1162-4. (26) Monchois, V.; Vincentelli, R.; Deregnaucourt, C.; Abergel, C.; Claverie, J. M. In Cell-Free Translation Systems; Spirin, A. S., Ed.; Springer: Berlin, 2002; pp 197-202. (27) Betton, J. M. Curr. Protein Pept. Sci. 2003, 4, 73-80.

demonstrate the potential for further applications, such as the high-throughput screening of drug candidates or enzymes. EXPERIMENTAL SECTION Materials. The RTS 100 Escherichia coli HY kit was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Fluorescein di-D-galactopyranoside (FDG) and phenylethyl D-thiogalactopyranoside (PETG) were purchased from Molecular Probes (Eugene, OR). Two pIVEX2.4 vectors containing the genes coding wt-GFP and β-galactosidase were obtained by the courtesy of Dr. Erhardt Fernholz, Roche Diagnostics GmbH (Penzberg, Germany). Radioactive L-[35S]methionine was obtained from (Amersham Biosciences, Amersham, U.K.). Mineral oil was purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany), and hybridization chambers for microarrays were from Scienion AG (Berlin, Germany). Technique. For the precision dispensing in nanoliter volume range, we have used a single-channel piezodispensing head with control unit form sciFLEXARRAYER (Scienion AG). The head consists of a glass capillary with an integrated piezoceramic. The piezonozzle with an inner diameter of 50 µm provides a droplet volume of 360 pL size. The volume range of 100-1000 nL was achieved through multiple droplet deposition using 100 Hz and applying a voltage of 100 V. All assays were performed in a 96-well glass microplate (Micronit BV, Enschede, The Netherlands), with a well volume of 1.5 µL. The well-to-well distance on the plate is 2.25 mm (Figure 1), which is identical to the distance used in standard 1536 plastic microplates. The fluorescent signal from the reaction in the wells was detected with a high-sensitivity laser scanner (a prototype developed by Perkin-Elmer). A single laser beam at 488 nm scans the glass microplate in the field of 20 × 20 mm2 containing 64 (8 × 8) wells. The resulting fluorescent signal at 535 nm is sent to a photomultiplier with photon counting. Methods. (1) Assessment of Protein Expression by SDSPAGE Using Radioactive L-[35S]Methionine. The in vitro transcription and translation mix (RTS 100 mix) was prepared as recommended by the manufacturer using a radioactive amino acid. Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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Components of the reaction were mixed and incubated for 1 h at 30 °C in the RTS ProteoMaster Instrument (Roche Diagnostics GmbH, Mannheim, Germany). Protein expression products were separated in 15% polyacrylamide gel with SDS (SDS-PAGE), and the radioactively labeled proteins were visualized by autoradiography using a PhosphoImager (Amersham Biosciences, Amersham, UK) and analyzed using ImageQuant 5.2 software (Amersham Biosciences). (2) In Vitro Expression of Green Fluorescent Protein (GFP) and β-Galactosidase from Batch. The RTS 100 mix was prepared in a 50-µL batch as recommended by the manufacturer and was incubated overnight in a 30 °C incubator without shaking. For the prediluted mix, the mix was prepared as described and diluted in 1× PBS prior to expression. Following protein expression, several dilutions were prepared using 1× PBS and dispensed using the piezonozzle at 100 V and 100 Hz for 5, 10, 15, and 20 s, so that the lowest volume was dispensed first. To avoid excessive evaporation, the samples were sealed with mineral oil after each time step. In the case of β-galactosidase, the fluorogenic substrate FDG was diluted in ddH2O to a concentration of 0.5 mM and dispensed for 5 s (corresponding to 180 nL) prior to the dispension of expression mix and was not sealed with mineral oil. (3) Expression and Detection of GFP and β-Galactosidase Activity in Nanowells. Two batches of RTS 100 mix were prepared for each protein, one with DNA as a positive control and one without DNA as a negative control. Additionally, a 1:10 dilution of the RTS 100 mix was prepared using 1× PBS for dilution. Prior to 30 °C incubation, different volumes of the dilutions were pipetted into the nanowells, using standard pipets (Gilson, P2), and sealed with mineral oil. For incubation, the nanowell plate was transferred into hybridization chambers, containing ddH2O to prevent evaporation, and placed in a 30 °C incubator overnight. In the case of β-galactosidase, the fluorogenic substrate FDG was diluted in ddH2O to a concentration of 5 µM and 0.2 µL was added starting with the lowest volume of the mix carrying the expressed β-galactosidase. (4) Inhibition Assay of β-Galactosidase Activity in Nanowells. The in vitro transcription and translation mix was prepared in a batch as recommended by the manufacturer and was incubated overnight in a 30 °C incubator without shaking. Following expression, the mix was diluted 1:10 in PBS and the inhibitor PETG was resuspended in ddH2O to a concentration of 33.3 mM. Different dilution steps of PETG were prepared, ranging from the original concentration of 333 µM to 333 nM. For the assay, 0.2 µL of inhibitor was pipetted into the nanowells followed by 0.6 µL of 1:10 diluted expression mix containing expressed β-galactosidase and 0.2 µL of 5 µM FDG. RESULTS AND DISCUSSION Determination of Detection Range of in Vitro Expressed Proteins. Two proteins, GFP and β-galactosidase, were expressed using a cell-free coupled in vitro transcription and translation system. The monomeric, active GFP has a chromophore formed by autocatalytic cyclization and subsequent oxidation enabling 1846 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

Figure 2. Undiluted cell-free expression of GFP. Top: GFP was expressed in a batch using an undiluted RTS 100 mix, and final dilutions of 1:10 and 1:100 were prepared. Different volumes of 720, 540, 360, and 180 nL were dispensed in duplicates into nanowells and scanned. Bottom: Diagram of signal intensity versus dispensed volume for both dilution steps.

fluorescent detection.28 β-Galactosidase consists of four identical subunits of 116.3 kDa forming a 465-kDa protein. The active β-galactosidase hydrolyzes sequentially its nonfluorescent substrate FDG to fluorescein monogalactoside and then to highly fluorescent fluorescein, which can be detected at a wavelength of 535 nm. Two 50-µL batches of both proteins were expressed in vitro, one incorporating L-[35S]methionine for radioactive labeling. GFP was also expressed using a 10-fold prediluted transcription and translation mix. The radioactively labeled batches were checked on an SDS-PAGE; the nonlabeled ones were used for analysis of the fluorescent spectra of GFP and β-galactosidase-processed FDG (data not shown). Dilution rows of expressed GFP derived from both, the undiluted as well as the prediluted mix, were prepared and transferred into the nanowells. The samples were overlaid with mineral oil to avoid evaporation and scanned with an excitation of 488 nm and an emission of 535 nm, using a highly sensitive laser scanner (Figures 2 and 3). Cell-free expressed GFP derived from the standard concentrated translation mix was detected in the 1:10 dilution but was hardly detected in the 1:100 dilution (Figure 2). GFP derived from the 10-fold prediluted translation mix was detected in the original 1:10 and in the 1:100 dilution of the standard translation mix (Figure 3). All dilution rows, with the exception of the 720-nL volume of the prediluted mix carrying GFP, displayed an increase of the signal intensity with increasing volume. A quantification of the signal was possible down to a volume of 180 nL (Figure 3). While both 1:10 dilutions displayed comparable signal intensities and amounts of active GFP, respectively, the amount of active GFP in the 1:100 prediluted translation mix was found to be higher. Reasons for that may be that synthesis of active GFP is dependent on spontaneous cyclization and subsequent oxidation. Nemetz and co-workers demonstrated that reactions with low amounts of oxygen result in accumulation of up to half of the improperly (28) Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12501-4.

Figure 3. Diluted cell-free expression of GFP. Top: A RTS 100 mix containing PT7-GFP-DNA was prepared and diluted 1:10 with PBS prior to incubation at 30 °C. Following expression, another dilution of 1:10 was prepared from the prediluted mix corresponding to a final dilution of the original mix of 1:100. Different volumes of 720, 540, 360, and 180 nL were dispensed in duplicate into nanowells and scanned. Bottom: Diagram of signal intensity versus dispensed volume for both dilution steps.

Figure 4. Undiluted cell-free expression of β-galactosidase. Top: β-Galactosidase was expressed in a batch using an undiluted RTS 100 mix, and a dilution of 1:1000 was prepared. A 180-nL aliquot of 0.5 mM FDG was dispensed into nanowells, and duplicates of the RTS 100 mix containing expressed β-galactosidase were added at volumes of 720, 540, 360, and 180 nL. Scanning was performed after several minutes of incubation at room temperature. Bottom: Diagram of signal intensity versus dispensed volume.

folded and therefore inactive GFP.29 Due to the low final concentration of GFP in the prediluted translation mix, the relation of oxygen to expressed GFP is probably higher in the prediluted mix than in the undiluted mix. This should allow formation of more active, fluorescent GFP. Dilutions of expressed β-galactosidase were prepared and the different samples were dispensed in volumes ranging from 180 to 720 nL into the nanowells, onto the predispensed substrate FDG (Figure 4). In vitro expressed β-galactosidase activity was detected successfully down to a volume of 180 nL of a 1:1000 dilution. In parallel, a negative control was performed, which displayed that the fluorescence created by FDG hydrolysis in the absence of β-galactosidase was negligible in this assay (data not shown). As shown for GFP, relative intensities of β-galactosidase-processed (29) Nemetz, C.; Reichhuber, R.; Schweizer, R.; Hloch, P.; Watzele, M. Electrophoresis 2001, 22, 966-9.

Figure 5. Undiluted cell-free expression of GFP in nanowells. Top: Two batches of an undiluted RTS 100 mix were prepared, one with PT7-GFP-DNA (A) and one lacking DNA (B). Volumes of 1000, 500, 250, and 100 nL from both batches were manually transferred into nanowells and sealed with 0.5 µL of mineral oil. The nanowells were incubated at 30 °C and scanned. Bottom: Diagram of signal intensity versus dispensed volume for both batches.

FDG increased proportionally with increased volume from 180 to 720 nL. Both in vitro synthesized proteins have been successfully detected in various dilutions and volumes, indicating a successful combination of cell-free expression and nanowell technology. Cell-Free Transcription and Translation in Nanowells. Batches of cell-free transcription and translation mix were prepared, and plasmid DNA carrying the coding sequences of GFP and β-galactosidase were added. For the expression of β-galactosidase, an additional 1:10 dilution of the translation mix was prepared. All samples were transferred in volumes ranging from 0.1 to 1 µL into the nanowells and were overlaid with mineral oil to prevent evaporation. Following 30 °C incubation for 16 h, substrate FDG was added in sequence from the lowest to the highest volume into the wells containing the expressed β-galactosidase. The nanowell plate was scanned with an excitation of 488 nm and an emission at 535 nm and the signals were quantified (Figures 5 and 6). Both, GFP and β-galactosidase were expressed successfully in volumes ranging from 0.1 to 1 µL. In addition, β-galactosidase was efficiently expressed using the prediluted translation mix (data not shown). These results show the feasibility of in vitro protein expression in a small-volume scale. The predilution of the translation mix is possible, since low amounts of expressed protein are sufficient for highly sensitive detection at nanoliter scale. Moreover, the predilution has the further advantage of reducing the required translation components and thereby costs. An additional advantage is that the dilution overcomes the incompatibility of many piezoelectric dispensers with highly viscous fluids, by the significant increase of fluidity by PBS. However, for the measurement of kinetics, upscaling of the single nozzle to a multidispenser system is required to allow the parallel addition of substrate and to avoid deviation of the results due to time differences caused by sequential dispension. To address the issue that other reactions might require the addition of further compounds or solutions, we performed in vitro Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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Figure 6. Undiluted cell-free expression of β-galactosidase in nanowells. Top: Two batches of an undiluted RTS 100 mix were prepared, one with PT7-β-galactosidase-DNA (A) and one lacking DNA (B). Volumes of 1000, 500, 250, and 100 nL from both batches were transferred into nanowells using the standard pipets and sealed with 0.5 µL of mineral oil. The nanowells were incubated at 30 °C, and 0.2 µL of 5 µM FDG was added before scanning. Bottom: Diagram of signal intensity versus dispensed volume for both batches.

expression directly in nanowells without an overlay of mineral oil using a hybridization chamber equipped with water reservoirs. This provides a high humidity within the closed systems and enables us to reduce evaporation of samples, resulting in successfully expressed GFP and β-galactosidase directly in nanowells (data not shown). Inhibition Assay of β-Galactosidase Activity in Nanowells. To show the usefulness of the nanowell format combined with the technology of cell-free protein synthesis, we performed an inhibition assay. In vitro expressed β-galactosidase was mixed with different dilutions of PETG, which is known to inhibit enzymatic activity of β-galactosidase.30,31 Following addition of the fluorogenic substrate FDG, different concentrations of PETG and the transcription and translation mix containing the expressed β-galactosidase were dispensed and we were able to monitor decreasing signal intensity with increasing PETG concentrations (Figure 7). Strong inhibition of β-galactosidase activity was achieved with PETG at final concentrations of 1-0.1% (v/v), whereas a final concentration of 0.001% (v/v) PETG resulted in comparable β-galactosidase activity levels as the noninhibited reaction. The increase of signal intensity with decreasing inhibitor concentration reflects the inhibition of β-galactosidase activity by PETG with an IC50 of 3.1 µM. Liquid Handling and Detection of Miniaturized Protein Assays. Miniaturization in the into nanoliter-volume range requires a nanodispensing method with a volume resolution of at least of 10 nL. Here, we have used a piezodispensing capillary with 360-pL single droplet size. Due to the well-defined geometry of the capillary and piezoceramic, the dispensed droplets are uniform at constant electronic parameters. However, the viscosity and consistency of the dispensed solution influence droplet formation and size. To ensure reliable droplet formation at different concentrations, we have increased the voltage from 63 to 100 V. (30) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-12. (31) Russo-Marie, F.; Roederer, M.; Sager, B.; Herzenberg, L. A.; Kaiser, D. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8194-8.

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Figure 7. Inhibition assay of cell-free expressed β-galactosidase in nanowells. Top: β-Galactosidase was expressed in a batch using an undiluted RTS 100 mix, and a dilution of 1:10 was prepared using 1× PBS. Concentrations of the inhibitor PETG were prepared ranging from 333 µM to 333 nM. For the assay, 200 nL of inhibitor was added to the nanowells, followed by 600 nL of the 1:10 diluted expression mix carrying expressed β-galactosidase and 200 nL of 5 µM FDG. Two assays were performed: the first one with duplicates of PBS, 66, 6.6, and 0.66 µM PETG (A) and the second one with duplicates of PBS, 6.6, 0.66, and 0.066 µM PETG (B) (both from left to right). Bottom: Diagram of signal intensity versus PETG concentration for both batches. The mean signal intensity was taken for the 6.6 µM and the 0.66 µM PETG concentration steps.

In preliminary experiments, we have analyzed different nanowell plate types (plastic, glass, silicon) for carrying out experiments in nanoliter scale (data not shown). On the basis of these results, we have chosen a miniaturized glass microplate with 96 wells ordered in a grid pattern of 8 × 12 with a pitch of 2.25 mm. This plate has several advantages compared to the other formats, such as the possibility of reusing the plate and the absence of background fluorescence. The 1.5-µL volume of the wells is optimal for experiments in higher nanoliter volume range (1001000 nL), and the glass microplate can be used in future standard miniaturized applications. However, the glass microplate used here has the limitation that the detection of very high intensity signals is problematic, since scattered light influences the measured signal intensities of adjacent wells. To prevent such effects, we have used different nanowell chips or distant wells on the same chip. Another possible solution to this problem may be the use of opaque microplates as already commonly used for fluorescent ELISA. The high-sensitivity laser scanner (excitation 488 nm, emission 535 nm) applied here has been used with low laser intensity to generate strong signals up to counting levels of 500 relative units with low background signals of 10 relative units. The scanning field of 20 × 20 µm enabled a parallel detection of 8 × 8 wells on the microplate. Additionally, we have scanned the microplate with a fluorescence camera (excitation 480 nm, emission, 540 nm; data not shown) and were able to detect signals with good resolution. This pilot study demonstrated the feasibility to perform cellfree protein expression and characterization in nanowell plates down to volumes of 100 nL. This format reduces the consumption of reagents required and allows the screening of large sets of samples. Moreover, screening against large protein expression libraries can be performed at reasonable cost, since consumption of cell-free transcription and translation reagents is reduced by a factor of 500. The dispensing of the reagents into the nanowells

can be automated, which improves the accuracy and efficiency of the system. The application of cell-free coupled transcription and translation systems allows the expression of toxic proteins and enhances yields for the expression of poorly expressible or insoluble proteins. The use of nanowell plates offers an improved sensitivity in comparison to standard microarray technology due to the exploitation of the third dimension, which increases the signal intensity per area.

proteins and their subsequent use in screening assays of different types of libraries (e.g., compounds, proteins). Moreover, large protein libraries can be generated in one array, by transfer of DNA samples coding for individual proteins to single wells, followed by dispensing of the translation mix. After incubation, such arrays can be applied to either functional assay or protein interaction screenings.

CONCLUSION We have shown the combination of nanowell chip technology and in vitro transcription and translation of proteins. Proteins have been dispensed, using different dispensing systems, or were directly expressed in vitro in nanowells to determine enzymatic activity. Additionally, we present an inhibition assay to indicate potential further applications, such as the high-throughput screening of drug candidates or enzymes with novel properties. Key to such screening experiments is the need for miniaturization to reduce costs, waste, and consumables. The combination of nanowell chip technology and cell-free expression of proteins allows the preparation of large batches of

ACKNOWLEDGMENT This work was founded by BMBF grant “Biofuture”, the BMBF project “Neue Anwendungspotentiale der in vitro-Proteinsynthese” (Team AB, Gruppe Nierhaus) and the Max-Planck-Society. D.J.C. gratefully acknowledges funding from the Health Education Authority and Science Foundation Ireland (SFI), Dublin 2, Ireland.

Received for review September 23, 2003. Accepted January 18, 2004. AC035114I

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