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Protein Expression and PuriWcation 36 (2004) 217–225 www.elsevier.com/locate/yprep

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems
T.V.S. Murthy, Weilin Wu,1 Q.Q. Qiu, Zhenwei Shi, Joshua LaBaer, and Leonardo Brizuela¤
Harvard Institute of Proteomics, 320 Charles street, Cambridge, MA 02141, USA Received 8 January 2004, and in revised form 26 March 2004 Available online 20 May 2004

Abstract Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18–159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially puriWed under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single aYnity puriWcation step from 50 l in vitro protein synthesis reactions. The in vitro protein expression plus puriWcation in a 96-well format and analysis of the proteins by SDS–PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step aYnity puriWcation oVers a rapid, eYcient alternative for the highthroughput screening of clones for protein expression and solubility. ? 2004 Elsevier Inc. All rights reserved.
Keywords: High-throughput protein expression; In vitro protein synthesis; Pseudomonas aeruginosa; Two-component system

The genome sequencing of several organisms and the automation of high-throughput cDNA and ORF cloning have facilitated the production of thousands of cloned genes in protein expression-ready formats [1–3]. Expression of proteins from these clones often necessitates the use of high-throughput expression systems, which are simple and can be used for expression of most of the targeted proteins with a minimum number of steps. The in vivo expression of proteins in bacteria is routinely used for high-throughput production of proteins due to its relative simplicity over other expression systems [4,5]. These high-throughput approaches were made possible by the availability of novel bacterial strains with highly inducible gene expression and eYcient plasmid vectors that couple the gene’s coding sequences with polypeptide tags that simplify puriWcaCorresponding author. Fax: 1-617-324-0824. E-mail address: lbrizuela@hms.harvard.edu (L. Brizuela). 1 Present address: Karolinska Institute, Center for Genomics and Bioinformatics, Berzelius Vag 25, 171 77 Stockholm, Sweden. 1046-5928/$ - see front matter ? 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.04.002
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tion and improve solubility [4–9]. Comparative studies of bacterial expression of proteins with various expression/puriWcation tags were previously reported from our laboratory [4] as well as others [5]. Although bacterial in vivo expression is widely used for high-throughput production of heterologous proteins, many of the steps in the process, such as transformation, synchronization of cultures, addition of the transcription inducing agents, cell lysis, and centrifugation/Wltration/separation steps, are time consuming and often require manual intervention during automation thereby reducing throughput. An alternative for proteomic expression of proteins is the use of cell-free (in vitro) systems. Proteins synthesized in the cell-free systems display the same accuracy as in in vivo translation [10]. Cell-free protein synthesis has several general advantages compared with in vivo expression, including the ability to express proteins that are toxic to the physiology of the host cells [11], the ability to incorporate non-natural amino acids [12–15], and the ability to rapidly express mutants of a protein for analysis [16].

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Cell-free protein expression involves the addition of template DNA containing the appropriate promoter elements to a bacterial extract that contains the relevant RNA polymerase and ribosomal machineries as well as the necessary substrates (tRNAs, amino acids, ATP, etc.), which then transcribes and translates the protein in vitro [17]. The process of making bacterial extracts and the reagents for protein synthesis is labor intensive. However, eVorts from several independent laboratories in the past few years led to the design of protocols to generate highly synthetic bacterial cell extracts capable of producing several hundreds of micrograms of protein per milliliter reaction volumes [16,18–21]. Protein synthesis can be prolonged by dialysis or a bi-layer diVusion system during the protein synthesis reaction, which simultaneously removes the toxic by-products and regenerates/reintroduces the reagents required for protein synthesis [19,22,23]. Bacterial cell-free systems have successfully expressed proteins that could not be expressed in vivo in bacterial and insect cells [24]. The cell-free protein synthesis also enables addition of detergents, chaperones, and appropriate ligands during protein synthesis, which may aid in proper folding of the proteins and shows promise for obtaining soluble and functional proteins [25]. Moreover, the use of a linearized DNA fragment as a template, such as that obtained by PCR, can eliminate the need for cloning/sub-cloning [26]. Despite numerous reports on the expression of discrete and large gene sets in bacterial in vitro and in vivo systems, none have compared the expression and puriWcation of the same set of proteins in the same plasmid vector background by both systems. In the present study, we have undertaken expression and puriWcation of a family of 63 response regulator proteins from Pseudomonas aeruginosa [27], using an N-terminal 6HIS tag. The molecular weights of these proteins range from 18 to 159 kDa. A comparison of in vivo and in vitro expression using the optimized conditions in our laboratory for both expression systems has been performed and is presented here. A schematic representation of the steps involved in the in vivo and in vitro protein expression and puriWcation procedures is shown in Fig. 1.

BrieXy, 150 ng of master clone (gene cloned into the plasmid vector, pDONR201) and 150 ng of pDEST17 (acceptor vector, Invitrogen) were recombined in a 10 l reaction with GATEWAY LR clonase reaction buVer and enzyme mix (Invitrogen). The reaction was allowed to proceed for 60 min at 25 °C and terminated by addition of 1 l proteinase K (Invitrogen) followed by incubation at 37 °C for 10 min. Two microliters of the reaction mix was transformed into DH5 competent cells. The cells after transformation were plated on an LB agar plate containing ampicillin (100 g/ml) and allowed to grow for 16 h at 37 °C. Single colonies were inoculated in liquid cultures, allowed to grow overnight, and stored as glycerol stocks. Five microliters of the stock was inoculated into 1 ml liquid cultures containing ampicillin, for subsequent plasmid DNA preparation. Preparation of bacterial cell extract The bacterial cell extract was prepared according to the protocols kindly provided by Dr. Yokoyama and are described elsewhere [12]. BrieXy, 3 L of BL21 codon plus RIL (Stratagene) was grown in LB medium containing 34 g/ml chloramphenicol, to an O.D600 of 2. Cells were harvested and washed twice with wash buVer A (60 mM potassium acetate; 10 mM Tris acetate, pH 8.2; 14 mM magnesium acetate; 2 mM DTT; and 7 mM 2-mercaptoethanol), and twice with wash buVer B (60 mM potassium acetate; 10 mM Tris acetate, pH 8.2; 14 mM magnesium acetate; and 2 mM DTT). The cell pellet was Wnally resuspended in 6.9 ml wash buVer B. 22.6 g of 0.17 mm diameter glass beads was added to the suspension and the cells were disrupted using the MSK B. Braun cell disruptor simultaneously maintaining a cold temperature with liquid CO2. The cell lysate was centrifuged at 30,000g and the supernatant was incubated with 0.3 volume of incubation buVer (300 mM Tris–Cl, pH 8.0, 9.2 mM Mg(OAc)2, 13.2 mM ATP, pH 8.0, 84 mM phosphoenolpyruvate, 4.4 mM dithiothreitol, 0.04 mM each amino acid, and 5 U pyruvate kinase (sigma)) at 37 °C for 80 min. The lysate was dialyzed 4 times 45 min each time against wash buVer B and Wnally centrifuged at 5000g for 10 min. The supernatant (S30 extract) was aliquoted in 96-well microtiter plates, snap-frozen in liquid nitrogen for 4 min, and stored at ?80 °C. The total protein concentration in the S30 extract is 10 mg/ml (determined by Bio-Rad protein assay reagent). In vitro protein expression and puriWcation Protein synthesis reaction was performed as described elsewhere [12]. Fifty microliters of reaction mixture contained 12 l S30 extract. The Wnal reaction mix contains 57 mM Hepes/KOH, pH 8.2, 1.2 mM ATP, 0.85 mM each of CTP, GTP, and UTP, 0.65 mM 3?, 5?-cyclic AMP, 35 g/ml folinic acid, 27.5 mM ammonium acetate,

Materials and methods Cloning and sub cloning The genes used for this study were obtained from our repositories of Pseudomonas aeruginosa gene collection (LaBaer et al., in revision). The clones were veriWed by end terminal sequencing (5? and 3?) of the genes in pDONR201 and comparing the sequencing results with the sequence in the Pseudomonas aeruginosa database. An LR reaction was performed to sub-clone the genes into the GATEWAY acceptor vector (pDEST17).

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Fig. 1. Schematic representation of 96-well protein expression and puriWcation by bacterial in vivo and in vitro methods.

195 mM potassium glutamate, 80 mM creatine phosphate, 250 g/ml creatine kinase, 1.8 mM dithiothreitol, 1.5 mM each of the 20 amino acids, 175 g/ml tRNA (Roche), 30 mM magnesium acetate, and ?1000 U/ml T7 RNA polymerase (prepared in-house according to protocols of Davanloo et al., [28]). Plasmid DNA for protein synthesis reaction was prepared by Qiaprep spin mini prep kit (Qiagen) and quantiWed by agarose gel analysis on a Chemidoc (Bio-Rad). Four hundred to Wve hundred nanograms of DNA was added to the reaction mix and the reaction was allowed to proceed for 2 h at 37 °C in a 96-well plate, with shaking at 300 rpm. For analysis of expression of protein in the total cell extracts after protein synthesis reaction, 2.0 l of the reaction was precipitated with 50 l cold acetone, air-dried, treated with 10 l of 1? SDS–sample buVer, heated for 5 min at

95 °C, and subjected to SDS–PAGE analysis. The gels were stained with biosafe Coomassie blue (Bio-Rad) for visualizing the bands. To determine the solubility of proteins, the cell-free reaction mixture after protein synthesis was subjected to centrifugation at 4500 rpm for 30 min at 4 °C. Two microliters of the supernatant was processed for SDS–PAGE analysis in a similar manner to that of the total cell-extract. For protein puriWcation, 50 l of the cell-free protein synthesis reaction containing the recombinant protein was diluted 1:2 with buVer A (100 mM NaH2PO4, 10 mM Tris–Cl, 8 M guanidine hydrochloride, pH 8.0) for denaturing conditions or buVer B (100 mM NaH2PO4, 500 mM NaCl, and EDTA-free protease inhibitors (Roche), pH 8.0) for non-denaturing conditions. The diluted sample was added to a 96-well Whatman GF/C

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plate aliquoted with 10 l Ni2+–NTA agarose beads (Qiagen) pre-washed with either buVer A or buVer B as needed. The suspension was allowed to rock for 15 min at room temperature, centrifuged, and washed twice (100 l each time) with wash buVer, WD (100 mM NaH2PO4, 10 mM Tris–Cl, 8 M urea, and 5 mM imidazole) for denaturing conditions or WN (100 mM NaH2PO4, 10 mM Tris–Cl, 500 mM NaCl, 5 mM imidazole, and EDTA-free protease inhibitors, pH 8.0) for non-denaturing conditions. The bound protein was eluted with 12.5 l elution buVer, ED (100 mM NaH2PO4, 10 mM Tris–Cl, 8 M urea, and 500 mM imidazole, pH 8.0) for denaturing conditions and EN (100 mM NaH2PO4, 10 mM Tris–Cl, 500 mM imidazole, and EDTA-free protease inhibitors, pH 8.0) for nondenaturing conditions. The eluate was treated with 12.5 l of 2? SDS sample buVer and heated for 5 min at 95 °C and the entire 25 l was subjected to SDS–PAGE. The gels were stained with Coomassie blue (Bio-Rad) for visualizing the bands. The presence of transmembrane helices or signal peptide in the proteins is determined using the software programs, TMHMM server v. 2.0 or Signal P v. 2.0, respectively (http://www.cbs.dtu.dk/services). Bacterial (in vivo) expression and puriWcation of proteins Cell growth, transformation, and protein puriWcation for in vivo production of proteins were performed according to the protocols in our laboratories and are described elsewhere [4]. BrieXy, BL21 star (DE3) pLysS transformants harboring the recombinant plasmids were grown at 37 °C as 1 ml cultures in a 96-well block (Marsh Biomedical products), to an O.D600 of ?0.7 and induced with 1 mM IPTG. After allowing a post-induction growth of 4 h, the cells were harvested, lysed with the lysis buVer (100 mM NaH2PO4, 10 mM Tris–HCl, 6 M guanidinium–HCl, 10% glycerol, and 10 mM 2-mercaptoethanaol, pH 8.0), and allowed to bind to Ni2+–NTA resin prewashed with lysis buVer. The resin with the bound protein was washed twice with wash buVer (100 mM NaH2PO4, 10 mM Tris–HCl, and 8 M urea, pH 8.0). The bound protein was eluted in 80 l elution buVer (wash buVer containing 500 mM imidazole, pH 8.0). Eluate (12.5 l) was treated with 12.5 l of 2? SDS–sample buVer and subjected to SDS–PAGE. The protein bands were visualized by staining with Coomassie blue. Western blot analysis Two microliters of total cell-free reaction after protein synthesis was incubated with 50 l ice-cold acetone for 5 min in a 96-well plate followed by centrifugation at 4500 rpm for 10 min. The supernatant was discarded, the pellets were allowed to air-dry and resuspended in 15 l SDS–sample buVer. For analysis of in vivo expression, 5 l of total culture after induction was treated with 5 l of 2? sample buVer. All the samples after treatment with

SDS–sample buVer were heated for 5 min at 95 °C, chilled on ice, and subjected to SDS–PAGE followed by Western blot analysis as described elsewhere [29]. BrieXy, the proteins were transferred onto a PVDF membrane and the membrane was blocked with 3% BSA for 1 h. The membrane was then probed with anti-tetra HIS antibodies (1:2000, Qiagen) for 2 h. The blot was washed thrice with PBST and incubated with HRP-linked antimouse secondary antibodies (1:5000, Amersham–Pharmacia) for 1 h. After three washes with PBST, the protein bands were detected by chemiluminescence.

Results Expression and puriWcation of 63 proteins in bacterial cell-free system Sixty-three genes from P. aeruginosa corresponding to response regulators from the 2-component system were tested for in vitro expression. N-terminal 6HIS tag was used based on previous results from our laboratory, which showed that 96-well one-step puriWcation under denaturing conditions provided reasonably pure recombinant protein preparations [4]. 51/63 tested proteins could be expressed and partially puriWed by Ni2+–NTA aYnity chromatography under denaturing conditions from 50 l of in vitro protein synthesis reactions (Fig. 2). The expression plus puriWcation in a 96-well format and SDS– PAGE analysis of these proteins in vitro were achieved in 4 h. The PA number and expected molecular weight of the proteins are shown in Table 1. Furthermore, a Western blot was also performed with a subset of 24 protein samples using anti-HIS antibodies (Fig. 3, Panel I-C). The molecular weight of the protein bands in the Western blot corresponded to the protein bands seen on the Coomassie gel (Fig. 3, Panels I-A and I-C). The protein yields varied from 100 ng to 1 g per 50 l reaction as determined by densitometric analysis. Most proteins with molecular weight greater than 90 kDa were not detected on the Coomassie gel after puriWcation (Fig. 2 and Table 1). To determine the solubility of the expressed proteins, the reaction mixture was subjected to centrifugation and the supernatant was analyzed by SDS–PAGE. The presence of soluble protein was scored by visualizing the protein band at the expected molecular weight on a Coomassie gel. 8/51 expressed proteins were detected in the soluble fraction and could be puriWed under nondenaturing conditions (Table 1). Comparison of expression and puriWcation of proteins in bacterial in vivo and in vitro systems To determine the expression pattern for the same set of proteins in vivo and in vitro, 24 proteins were subjected to bacterial in vivo and in vitro expression and

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Fig. 2. SDS–PAGE analysis of 63 proteins from P. aeruginosa aYnity puriWed under denaturing conditions. Fifty microliters of cell-free reaction mixture after protein synthesis was subjected to Ni2+–NTA aYnity puriWcation under denaturing conditions and the entire eluate was analyzed by SDS–PAGE. The puriWed recombinant protein is indicated with a black diamond on the left side of the protein band.

puriWcation. The total reaction mixture after in vitro protein synthesis and total bacterial culture following IPTG induction for in vivo expression were analyzed by SDS–PAGE and Coomassie staining as shown in Fig. 3 (Panel I-A and II-A). The apparent ratio of expressed protein/total protein appears to be several fold higher for in vivo expression than in vitro expression (Fig. 3, Panels I and II, A). Single-step Ni2+–NTA aYnity-puriWed protein obtained from either 200 l of in vivo bacterial culture or 50 l of in vitro extract under denaturing conditions is also shown in Fig. 3 (Panels I-B and II-B). The same 22/ 24 proteins were detected at the expected molecular weight for both systems, and one of the gene products, PA3204, appeared as a truncated product in both. Although the expressed proteins appeared as dominant bands of the expected size, some also displayed products of lower molecular weight for both in vitro and in vivo expression in the Western blot analysis (Fig. 3, Panels IC and II-C). The protein encoded by the gene, PA2479, showed relatively better expression in vitro compared to

in vivo expression. Two of the gene products (PA0034 and PA3204) are barely visible in the in vitro system due to low expression levels (Fig. 3, Panel I-A and I-B), but could be detected in the in vivo expression systems as well as in Western blots of in vivo and in vitro expression systems (Fig. 3, Panels I-C, II-A, II-B, and II-C).

Discussion In this study, we have used a bacterial cell-free system to express and purify 63 P. aeruginosa response regulator proteins using 50 l reactions in a 96-well format. Furthermore, we have expressed and partially puriWed a subset of this gene set using a high-throughput cellbased bacterial expression to compare the yield and purity obtained using these two systems. In the in vitro system, 51/63 tested proteins were detected on Coomassie blue-stained gels whereas 10/63 proteins were either absent or not detectable presumably due to low levels of expression (050 ng after single-step Ni2+–NTA aYnity

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T.V.S. Murthy et al. / Protein Expression and PuriWcation 36 (2004) 217–225 Table 1 (continued ) PA0928?? PA2583? PA4112?? PA4656 106 114 159 39 + + ? ? ? ? ? ?

Table 1 List of proteins of Pseudomonas aeruginosa, expressed in bacterial cellfree system Gene name Expected Mol. Wt (kDa) PuriWcation under denaturing conditions + + + + ? + + + + + + + + + + + + + + + + + + + + + ? + + + + + + ? + + ? + + + + + + + + + + + + + + + ? + + + ? ? ? Solubility/ puriWcation under non-denaturing conditions + + + ? ? ? ? ? ? ? ? ? + ? ? + + + ? ? ? ? ? ? ? ? ? ? ? + ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

PA0179 PA1456 PA4493 PA3045? PA0034 PA3948 PA3714? PA2586 PA0601 PA1397 PA4196 PA4776 PA3879 PA3604 PA1978 PA2657 PA4381 PA3204* PA1437 PA1179 PA3077 PA0756 PA0463 PA2479 PA2523 PA1980 PA1157 PA1799 PA4032 PA3192 PA0929 PA4101 PA5200 PA4296 PA5364 PA2881 PA2686 PA3702 PA0173 PA4396 PA3947 PA4781* PA2798 PA1335 PA4547 PA2572 PA5511 PA5483 PA5166 PA1099 PA4726 PA5125 PA4843 PA3846 PA1611?? PA2177 PA2824?? PA1243 PA3462??

18 19 26 27 28 28 28 28 28 28 29 29 29 28 29 30 30 30 31 31 30 29 31 30 31 30 31 31 32 32 32 33 33 36 37 39 39 43 42 45 48 49 48 52 55 56 55 54 56 56 57 58 67 26 76 82 91 98 107

The PA number refers to the Pseudomonas aeruginosa gene ID and is shown in column 1. The molecular weight of the proteins in kilodaltons is shown in column 2. Proteins which showed successful puriWcation are denoted by a plus (+) and proteins which failed to show expression are denoted by a minus (?). The success of puriWcation of proteins is determined by visualizing a protein band at the expected molecular weight, on a Coomassie gel, after puriWcation under denaturing conditions (column 3) or non-denaturing conditions (column 4). Proteins which appear as a major band after puriWcation under denaturing conditions but do not correspond with the expected molecular weight are represented with an asterisk (¤) after the PA number. A black dot (?) or black diamond (?) after the PA number indicates proteins with predicted transmembrane helices or signal peptide, respectively.

puriWcation under denaturing conditions). Forty nine out of 51 expressed proteins showed a major protein band at the expected size. In addition to the 49 proteins, two samples displayed proteins of lower molecular weight. The appearance of protein products with molecular weights lower than expected in some of the lanes suggests degradation or translational pausing (Fig. 3, Panel I-C), which was previously observed in bacterial cell-free expression [30]. We attempted to avoid this problem by expressing the same set of 63 proteins with a C-terminal 6HIS tag (which allows puriWcation of only full-length protein) and purifying them under denaturing conditions. Most proteins with the C-terminal 6HIS tag either failed to express or showed poor expression levels in both in vivo and in vitro expression systems (data not shown). For the proteins used in this study, the presence of amino terminal 6HIS tag dramatically enhanced the expression, perhaps the region around the start of translation being better optimized. EVect of the location of the aYnity tag on the expression of proteins in cell-free systems was previously reported [31]. In general, the ratio of target protein/total protein appeared to be higher for in vivo expression than in vitro expression (Fig. 3, Panel I-A and II-A). The Wnal protein concentration of the bacterial extract in the in vitro protein synthesis reaction (?2.5 g/ l) is higher compared to the total protein concentration of the lysed bacterial cells (?0.25 g/ l). This results in higher proportion of contaminating host proteins in the cell-free system after single-step aYnity puriWcation in the 96-well format (as seen in Figs. 2 and 3). Use of two aYnity puriWcation tags may be helpful to achieve full-length proteins with higher purity and overcome the above problems. For the same set of genes, the cell-free system produced less protein than for expression in vivo (see Fig. 3). Nevertheless, the speed and yields provided by the bacterial extracts are more than adequate for assessing the

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Fig. 3. Comparison of in vivo and in vitro expression of 24 proteins of P. aeruginosa. The expression (A), Ni2+–NTA aYnity puriWcation under denaturing conditions (B), and Western blot analysis (C) for in vitro expression (Panel I) and in vivo expression (Panel II) are shown. For expression, 2 l of in vitro synthesis reaction mix or 5 l of in vivo induced culture is subjected to SDS–PAGE analysis. For Ni2+–NTA puriWcation under denaturing conditions, puriWed protein obtained from 200 l of in vivo culture or 50 l of in vitro synthesis was analyzed by SDS–PAGE. Western blot analysis was performed with 5 l of in vivo cultures or 2 l of in vitro synthesis reactions as described.

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relative expression and solubility of the proteins encoded by large collections or libraries of genes. Furthermore, miniaturization of assay conditions for high-throughput biochemical/immunological studies, combined with the use of methods for arraying of proteins on glass slides, may obviate the requirement for higher yields of proteins [32,33]. Conversely, doubling the reaction volumes of in vitro protein synthesis reactions showed a corresponding increase in the amounts of protein without the need for dialysis during the protein synthesis reaction (data not shown). For the set of proteins in this study, only 8/63 proteins were soluble and could be recovered under non-denaturing conditions from the in vitro system. Among the Wrst 24 proteins listed in Table 1, seven proteins could be puriWed from the in vitro expressed set under non-denaturing conditions. All of these but one (PA2657) could be puriWed from the in vivo expressed set under nondenaturing conditions, indicating a signiWcant overlap of soluble proteins between the two systems (data not shown). In addition, PA3948 was puriWed from the in vivo expression but not from the in vitro expression system under non-denaturing conditions. Low percentage of solubility of 6HIS tagged proteins was previously reported for diVerent gene sets [4,34]. Increasing the applicability of the bacterial cell-free system for functional proteomics may beneWt from high-throughput methods to renature the denatured proteins or the addition of chaperones/detergents to the in vitro protein synthesis reactions to ensure proper folding of the recombinant protein. In addition, the use of alternative tags when possible may also enhance expression and solubility. Expression of 16 proteins from Homo sapiens and ten proteins from Dengue 2 and West-Nile viruses in the bacterial in vitro system in the 96-well format showed similar results to those discussed above, in terms of expression and puriWcation, and indicates that the bacterial cell-free system can be used for high-throughput protein production across species (data not shown). The steps involved in high-throughput expression and puriWcation of proteins in in vivo and in vitro systems are illustrated in Fig. 1. Given the simplicity with which proteins can be expressed from the pre-made extracts, in vitro expression oVers a dramatic increase in throughput. However, production of bacterial cell extracts with higher synthetic capacity plus optimization of the in vitro transcription/translation reaction is required for applications such as structural genomics, where higher yield and purity of protein are needed. In conclusion, comparative analysis of the expression of P. aeruginosa proteins in vitro and in vivo suggests that the bacterial cell-free system is an eYcient way to express microgram amounts of proteins in a high-throughput format, and that high-throughput expression, puriWcation, and analysis of proteins greatly beneWts from the use of the cell-free system. On the other hand, high-throughput

expression of proteins in bacteria provides higher protein yields and purer protein preparations, but at a higher cost in terms of time and process complexity (Fig. 1).

Acknowledgments We thank Dr. S. Yokoyama for kindly providing the protocols for preparation of bacterial cell-free extracts. We thank all the members of the Harvard Institute of Proteomics for their constant support. We thank Dr. Steve Lory for his help in selecting the genes of the two-component system. This work was funded by a Cystic Fibrosis Foundation grant.

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