Short Technical Reports A quick in vitro pathway from prokaryotic genomic libraries to enzyme discovery
MATERIALS AND METHODS
Lyubov A. Ryabova1,2, Sabrina Guillemer1, Stéphanie Pallas1, Cécile Persillon1, Fabrice Lefèvre1, Jean-Michel Masson1,3, and Gilles Ravot1
Unless otherwise stated, all reagents are from Sigma-Aldrich, Saint Quentin Fallavier, France. Pseudoalteromonas sp. (P1734; genome size about 1500 kb) is a strain from the exclusive biodiversity collection of microorganisms that has been isolated from marine environments (Protéus, Nîmes, France). Strains are available through partnership with Protéus. The Escherichia coli strain MC1061 Rec A was used as a recipient host for amplification of the genomic library of Pseudoalteromonas sp. Bacterial S30 cell-free extracts were prepared from E. coli strain MC1061 DE3 carrying a plasmid encoding either T7 or T3 RNA polymerase in a procedure described by Zubay (12).
SA, Nîmes, 2Institut de Biologie Moléculaire des Plantes, laboratoire propre du Centre national de la recherche scientifique (CNRS), conventionné avec l’Université Louis Pasteur, Strasbourg Cédex and 3Institut National des Sciences Appliquées (INSA), Toulouse Cédex, France BioTechniques 45:63-68 (July 2008) doi 10.2144/000112820
Screening of prokaryotic genomes in order to identify enzymes with a desired catalytic activity can be performed in vivo in bacterial cells. We propose a strategy of in vitro expression screening of large prokaryotic genomic libraries based on Escherichia coli cell-free transcription-translation systems. Because cell-based expression may be limited by poor yield or protein misfolding, cell-free expression systems may be advantageous in permitting a more comprehensive screen under conditions optimized for the desired enzyme activity. However, monocistronic messages with an improved leader initiation context are typically used for protein production in vitro. Here, we describe successful use of a Pseudoalteromonas genomic DNA library for in vitro expression of DNA fragments carrying multiple open reading frames (ORFs) in the context of their authentic translation initiation sites and regulatory regions. We show that ORFs located far from the 5′′ and 3′′ ends of polycistronic transcripts can be expressed at a sufficient level in an in vitro transcription-translation system in order to allow functional screening. We demonstrate the overall cell-free functional screen strategy with the successful selection of an esterase from Pseudoalteromonas.
INTRODUCTION During the last decade great improvements have been made in largescale cell-free translation as well as in producing correctly folded proteins in vitro (1–4). The application of cellfree systems allows the expression of toxic proteins and poorly expressible or insoluble proteins. Prokaryotic and eukaryotic in vitro protein synthesis systems are already widely used for rapid production and analysis of protein mutants, for incorporation of unnatural amino acids for structural studies, and for carrying out molecular selection and evolution in vitro (for review, see Reference 5). For example, recent in vitro studies describe cell-free systems for parallel expression of up to 100 coding regions cloned under various phage RNA polymerase promoters (6,7). These strategies have mainly used PCR amplification; however, PCR requires some sequence information about target genes and often fails to amplify through GC-rich regions in long DNA fragments (8,9). So far, Vol. 45ı No. 1 ı 2008
there have been no reports of direct prokaryotic genome expression for screening purposes. If successful, this would offer an additional screening tool for discovery of cytotoxic, poorly expressed, and unstable proteins. In vitro protein synthesizing machinery has been successfully used with DNA templates carrying multiple ribosomal protein genes to study posttranscriptional feedback regulation in ribosomal protein synthesis (10,11), suggesting that prokaryotic genomic fragments carrying two or more open reading frames (ORFs) using their authentic translation initiation start sites can be successfully expressed in vitro. Here, we describe a high-throughput cell-free functional screen (CFFS) to identify a desired functional activity from a Pseudoalteromonas genomic DNA library, thus demonstrating that large genomic fragments can be expressed in vitro. We believe that this in vitro method can complement wellknown in vivo screening methods when proteins that are difficult to express have to be analyzed.
Bacterial Strains and S30 Extracts
Construction of a Pseudoalteromonas Genomic DNA Library A plasmid library was constructed by ligation of genomic DNA, partially digested by Sau3AI, into the BamHI site of pBSKSII+, which is flanked 5′ and 3′ by T7 and T3 promoters, respectively. The size of cloned inserts ranged from 3 to 4 kb. The plasmid library was amplified by transformation of E. coli MC1061 Rec A strain (56,000 clones). Isolated E. coli colonies were selected and a CCS Packard Automate liquid handling system (Perkin Elmer, Courtaboeuf, France) was used to inoculate wells of 96-well plates (Costar 3897, Corning-Costar Corp., Cambridge, MA, USA) containing 150 μl of LB. DNA Amplification and Extraction for in vitro Tests and Library Screening Cultures were grown overnight at 37°C with vigorous agitation in 96-well plates. Using a CCS Packard Automate, plates with cell cultures were centrifuged at 2000× g, 4°C, for 5 min. The pellet in each well was resuspended in 150 μL of lysis buffer (Hepes-KOH pH 7.6, 0.1 mM EDTA, 4% sucrose, 0.5% Triton X100, and 0.1 mg/mL lysozyme) and incubated at 95°C for 30 www.biotechniques.com ı BioTechniques ı 63
Short Technical Reports
min and then at 4°C for 30 min. After centrifugation of plates at 2000× g for 10 min, 10 μL lysis supernatant was transferred to a fresh microtiter plate. These plates were either used directly for transcription-translation reactions or frozen at -20°C until processed for further screening steps.
Cell-free Transcription-Translation System Coupled transcription-translation reactions were prepared according to Kim and Choi (13) with minor modifications. Transcription-translation reactions (30 μl) were set up in a 96well format in a solution containing lysis supernatant (30% v/v), 55 mM Hepes-KOH (pH 7.6), 4 mM NTPs (ATP, CTP, GTP, and UTP), 0.6 mM cAMP, 34 μg/ mL folinic acid, 0.2 mM EDTA, 0.17 mg/ mL tRNA (Roche Diagnostics, Meylan, France), 3% PEG-8000, 24 mM Mg(OAc)2, 36 mM NH4Ac, 200 mM K glutamate, 0.25 mM all amino acids, 5 mM DTT (MP Biomedicals, Illkirch-Graffenstaden, France), 0.2 mM spermidine, 60 mM acetyl phosphate, and 20% S30 extract (300–500 A260). Rifampicin (4 μg/mL) was added in the reaction to prevent use of E. coli promoters. Reactions were incubated at 30°C for 3 h, after which time components of the esterase test were added or SDS-PAGE analysis was performed. The same reaction setup was used for transcription-translation of isolated DNAs. For in vitro transcription-translation reactions using [35S]methionine, the 0.5 mM amino acid mixture was replaced by 0.25 mM amino acids without methionine, together with 0.2 μΜ [35S]methionine (1175 Ci/mmol) (Perkin Elmer, Villebon sur Yvette, France). Esterase Activity Test C2-CLIPS-O [2-hydroxy-4-( [2-hydroxy-4-(p nitrophenoxy) butylacetate] substrate was synthesized from 4-bromobutene as described for the corresponding fluorescent umbelliferone derivatives (14,15). The test mixture, containing 160 mM PIPES buffer at pH 7.0, 1.7 mM C2-CLIPS-O substrate, and 64 ı BioTechniques ı www.biotechniques.com
In vitro transcription-translation
Figure 1. In vitro expression screening of a Pseudoalteromonas genomic library for esterase activity. (A) Schematic representation of the cell-free functional screen (CFFS) strategy. (B) A sample of genomic fragments from the library amplified and isolated from cellular extracts in 96-well format. The top panel shows agarose gel analysis of untreated lysis supernatant (LS), while the bottom panel shows PCR amplification of lysis supernatant using T3 and T7 primers. (C) Esterase-positive clones detected by CFFS in 96-well plates following transcription-translation reactions in LSs of E. coli expressing a Pseudoalteromonas genomic library. The mean OD414 (ΔODmean) was calculated for each microtiter plate. This mean value was then subtracted from all the ΔOD414 values in the plate. Esterase activity was detected in plate MP06, well F11 (MP06F11), and in plate MP29 well B5 (MP29B5). (D) Analysis of esterase activity resulting from the esterase-positive clones MP06F11 (lanes 1, 2), MP29B5 (lanes 3, 4), and a green fluorescent protein (GFP)-encoding plasmid (lanes 5, 6) retested in a cell-free transcriptiontranslation system initiated with 30% lysis supernatant (lanes 1, 3, 5) or 40% lysis supernatant (lanes 2, 4, 6). Data from three experiments.
the sample (representing 11% of the mixture volume), was incubated at 35°C for 2 h. Samples were cooled on ice, and 1 mg/mL BSA (BSA), 28 mM NaIO4, and 45 mM Na2CO3 were added to the mixture. After 10 min of incubation at room temperature, samples were centrifuged at 16,000× g for 5 min, and the supernatants were used for OD measurements. The OD of the yellow p-nitrophenol product was recorded at λ414nm using a Spectramax
190 microplate spectrophotometer (Molecular Devices, St. Grégoire, France). Activities expressed at OD414 were compared with a negative control. SDS-PAGE Gel Analysis For gel analysis of translation products labeled with [35S]methionine, aliquots from translation reactions were mixed with SDS-sample buffer, boiled for 2 min, and applied to a 10% SDSVol. 45 ı No. 1 ı 2008
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Figure 2. Multiple start sites within a genomic fragment are accessible to prokaryotic ribosomes in vitro. (A) Schematic representation of the Pseudoalteromonas sp. genomic fragment gfr1 showing the relative positions of four open reading frames (ORFs) encoding a putative transcriptional regulator (TrR), glutamate synthase (GluS), esterase (Est), and rotamase (Rot). gfr2 and gfr3 represent the truncated versions of gfr1 found in clones MP29B5 and MP06F11, respectively. Numbers represent nucleotide positions from the 5′ end of the clone. (B) Sequences of putative ribosomal binding sites 15 nt upstream of each ORF present in gfr1. The start codon of each ORF is highlighted. Elements of the Shine-Dalgarno region are underlined. An additional putative small ORF in front of the TrR gene is italicized. (C) [35S]-labeled proteins synthesized in transcription-translation reactions initiated with 2.9 μg of gfr1 (lane 1), 1.2 μg of gfr2 (lane 2), 1.2 μg of gfr3 (lane 3), or without added DNA (lane 4) were visualized by autoradiography following electrophoresis in a 12% SDS-PAGE gel. (D) Comparison of functional esterase activity of polypeptides synthesized in transcription-translation reactions initiated with 2.9 or 5.8 μg of plasmidencoding green fluorescent protein (GFP) (lanes 1 and 2), 2.9 or 5.8 μg of gfr1 (lanes 3 and 4), 1.2 or 2.4 μg of gfr2 (lanes 5 and 6), and 1.2 or 2.4 μg of gfr3 (lanes 7 and 8). Data from five experiments.
polyacrylamide gel. Gels were fixed in 10% methanol, 7.5% acetic acid, and exposed to Kodak Biomax MR film (VWR, Limonest, France) at -70°C. RESULTS AND DISCUSSION We have devised a cell-free functional screen based on in vitro expression of prokaryotic genomic libraries that enables us to search for desired enzyme activities. Figure 1A illustrates the four main steps of our CFFS screening strategy: (i) generation of a prokaryotic genomic library; (ii) bacterial amplification of DNA 66 ı BioTechniques ı www.biotechniques.com
fragments; (iii) expression of these fragments in a cell-free transcriptiontranslation system; and (iv) identification of enzymatic activities using a functional test. As esterases are enzymes of high value for industrial and pharmaceutical production processes, we focused on identifying genomic fragments that encode proteins with an esterase activity. First, we tested each step of our strategy using a recombinant plasmid (pBSKS-T3-est) encoding an esterase homologous to Acinetobacter lwoffii 16C-1 esterase (16) isolated from the Pseudoalteromonas library by a hybridization colony blot approach.
E. coli colonies transformed with pBSKS-T3-est were used to inoculate a 96-well culture plate. After amplification, cells were disrupted and cell debris precipitated. Lysis supernatants were used for transcription-translation. To maximize the efficiency of the cellfree transcription-translation reactions using lysis supernatant as a source of recombinant DNA, several parameters had to be optimized, including S30 extract, which is the most expensive and time-consuming component of this assay (the lowest concentration suitable to generate a strong signal and, thus, a large signal window, was chosen; see Supplementary Figure S1A available online at www.BioTechniques.com). Using optimized conditions we demonstrated successful detection of esterase activity in the well corresponding to pBSKS-T3-est but not in the remaining wells (Supplementary Figure S1B). Next, a Pseudoalteromonas DNA library was screened for esterase activity using the CFFS system developed as described above. A total of 5000 clones were grown in duplicate using 100 microtiter plates and an automated compound delivery robot (CCS Packard Automate). After overnight incubation, MC1061 Rec A transformants harboring recombinant plasmids were lysed and cell debris was precipitated. Agarose gel analysis of lysis supernatants from 13 randomly chosen wells from one 96-well plate is shown in Figure 1B (top panel). PCR amplification of these lysis supernatants using T3 and T7 primers indicates the range of insert sizes (Figure 1B, bottom panel). Lysis supernatants that had been clarified by centrifugation were directly added to coupled cell-free transcriptiontranslation reactions in matching wells of two fresh 96-well plates. Each clone of the library was treated in duplicate and expressed in the cell-free system with either T7- or T3-RNApolymerase-containing S30 extract. Following CFFS and testing for esterase activity using a fluorogenic assay, 10 possible hits [relative ΔOD414nm units >0.05] were identified (representative results obtained with two microtiter plates, MP06 and MP29, are presented in Figure 1C) and retested in duplicate to ensure that the initial Vol. 45 ı No. 1 ı 2008
result was truly positive in a second in vitro test with lysis supernatant (data not shown). Finally, two positive clones, MP06F11 and MP29B5, were confirmed. The other putative positive clones were all found in either the first or the last row of the plate (mainly A1 or H1 coordinates) and represented false positives, due to edge effects (all are located on the periphery of the microtiter plate). Quantification of functional esterase activity resulting from the esterase-positive clones MP06F11 and MP29B5, retested in a cell-free transcription-translation system initiated with 30% or 40% lysis supernatant, are summarized in Figure 1D together with the results obtained from a plasmid encoding the green fluorescent protein (GFP) as a negative control. The functional esterase activities, together with the expected size of synthesized products (45 kDa) led us to conclude that we have successfully identified two esterase-positive clones from the Pseudoalteromonas DNA library. The two positive clones, MP29B5 and MP06F11, were sequenced and both found to encode an esterase homologous to Acinetobacter lwoffii 16C-1 esterase. In general, prokaryotic operons are long and contain several ORFs preceded (or not) by Shine-Dalgarno regions, and some ORFs even overlap (17,18). Success of CFFS depends on the ability of the cell-free transcription-translation system to express long genomic fragments encoding multiple successive ORFs. When we used sequences from our esterase-positive clones to probe our Pseudoalteromonas DNA library by a hybridization colony blot approach, we isolated a 7.2 kb fragment. This fragment, which we called gfr1, contains four long ORFs flanked by 5′- and 3′untranslated regions (UTRs) of 546 and 745 nucleotides in length, respectively (Figure 2A). The first ORF encodes a 259-amino acid protein homologous to a transcriptional regulator (TrR); the second encodes glutamate synthase (GluS), a protein 261 amino acids long; the third a 365-amino-acid esterase (Est); and the last ORF encodes rotamase (Rot), a 181-amino-acid protein. Analysis of potential Shine-Dalgarno regions in front of each ORF reveals short purine-containing sequences
located 15 nt upstream of each start site (Figure 2B). Our two positive clones, MP29B5 and MP06F11, were found to correspond to the 3′ end of gfr1 (these clones were denoted gfr2 and gfr3, respectively; Figure 2A). We analyzed the accessibility of the start site of the esterase ORF within the very long genomic fragment in gfr1, as well as in gfr2 and gfr3, in our in vitro transcription-translation system. The ORFs of grf1, gfr2, and gfr3 were expressed in cell-free transcriptiontranslation reactions in the presence of l-[35S]methionine. The results demonstrated that all ORFs on each construct are expressed with relatively similar efficiencies (peptides encoded by the TrR, GluS, Est, and Rot ORFs contain 8, 5, 4, and 9 Met residues, respectively), with the level of expression of esterase being somewhat higher (the band labeled ΔEst1 apparently represents incomplete esterase proteins; Figure 2C). The rotamase coding region contains two internal AUGs 109 and 127 nucleotides downstream of the rotamase start site. These two additional start sites may explain the presence of the shorter polypeptide bands on the protein gel (Figure 2C). Expression of esterase from the long genomic fragment, gfr1, where the esterase ORF is located 4676 nt downstream of the 5′ end, and is preceded by two other ORFs, indicates the ability of ribosomes to initiate internally on this long genomic fragment. In gfr2 and gfr3, the esterase gene is located 709 or 51 nucleotides downstream of the T7 promoter, respectively; no significant effect of the longer 5′-UTR on esterase production in vitro was detected (Figure 2C, cf. lanes 2 and 3). Duplicate transcription-translation reaction mixtures (without l[35S]methionine) were used for analysis of esterase activities of the synthesized polypeptides (Figure 2D). The functional yield of in vitro-expressed esterases from gfr2 and gfr3 was found to be similarly high, again showing that the level of protein expression was not significantly affected by the distance between the first ORF and the transcription start site. The level of esterase activity resulting from transcription-translation of gfr1 (4674 nt downstream of the 5′ end and preceded by two other ORFs) was about
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15% of that attributable to expression of gfr3 (51 nt 5′-UTR), thus confirming that the esterase start site within the long genomic fragment was well recognized in vitro. The CFFS methodology allowed us to screen a library of >0.5 × 104 different variants in less than a day. The throughput of the method could be significantly increased by combining the robotic system with automated compound delivery and a 384-well plate format. This technology is highly compatible with use of a plasmid miniprep 96well kit to purify genomic fragments. The replacement of lysis supernatant by plasmid preparations would allow a significant increase of DNA concentration in the transcription-translation reaction, which should increase the level of protein production by up to 10-fold. To decrease the number of screening reactions, we tested the possibility of expressing a pool of genomic fragments in vitro in a single reaction and showed that the cell-free system charged with up to 10 genomic fragments (obtained from 10 pooled colonies) was able to express functionally active polypeptides (data not shown). In vitro functional screening methods such as ribosomal display (for a comprehensive review of this and other in vitro methods see Reference 19), have been already proposed, but these require modification of the synthesized protein in order to physically attach it to its gene. We propose direct expression of genome fragments containing two or more ORFs with their authentic translation initiation sites and regulatory untranslated regions in cell-free expression systems for screening purposes. CFFS has the following additional advantages: (i) the microplate compartmentalization method links a specific genotype with its corresponding phenotype without any modification of the protein synthesized (20) and allows for immediate in situ functional testing of enzymatic activity in vitro and (ii) cell-free expression of a genomic library should allow toxic, unstable, and aggregation-prone proteins to be synthesized. But, of course, an appropriate and sensitive functional test applicable to in vitro synthesized proteins is required. In summary, the CFFS method developed here will be a valuable tool 68 ı BioTechniques ı www.biotechniques.com
to complement cell-based discovery techniques toward the goal of screening the potentially vast pool of enzymes available in nature to carry out reactions on a wide range of natural and synthetic substances. ACKNOWLEDGMENTS
We are grateful to Joel Querellou (Institut Français de Recherche pour l’Exploitation de la Mer) for Pseudoalteromonas strain P1734 and to Elisabeth Senaux for excellent technical assistance. COMPETING INTERESTS STATEMENT
The authors declare no competing interests. REFERENCES 1. Spirin, A.S., V.I. Baranov, L.A. Ryabova, S.Y. Ovodov, and Y.B. Alakhov. 1988. Continuous cell free translation system capable of producing polypeptides in high yield. Science 242:1162-1164. 2. Kim, T.W., D.M. Kim, and C.Y. Choi. 2006. Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. J. Biotechnol. 124:373-380. 3. Ryabova, L.A., D. Desplancq, A.S. Spirin, and A. Plückthun. 1997. Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat. Biotechnol. 15:79-84. 4. Kawasaki, T., M.D. Gouda, T. Sawasaki, K. Takai, and Y. Endo. 2003. Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. Eur. J. Biochem. 270:4780-4786. 5. Jermutus, L., L.A. Ryabova, and A. Plückthun. 1998. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9:534-548. 6. Graentzdoerffer, A. and C. Nemetz. 2003. High-throughput expression-PCR using universal plasmid-specific primers. BioTechniques 34:256-260. 7. Busso, D., R. Kim, and S.H. Kim. 2004. Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins. J. Struct. Funct. Genomics 5:69-74. 8. Agarwal, R.K. and A. Perl. 1993. PCR amplification of highly GC-rich DNA template after denaturation by NaOH. Nucleic Acids Res. 21:5283-5284. 9. Varadaraj, K. and D. Skinner. 1994. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich
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Received 7 October 2007; accepted 2 December 2008. Address correspondence to Lyubov A. Ryabova, Institut de Biologie Moléculaire des Plantes, laboratoire propre du CNRS (UPR 2357) conventionné avec l’Université Louis Pasteur (Strasbourg 1), 12 rue du Général Zimmer, 67084 Strasbourg Cédex, France; e-mail: [email protected]
or Gilles Ravot, Protéus SA, 70 allée Graham Bell, Parc Georges Besse, 30000 Nîmes, France; e-mail: [email protected]
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