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A New Reporter Gene System Suited for Cell-Free Protein Synthesis and High-Throughput Screening in Small Reaction Volumes
Analytical Biochemistry 297, 177–182 (2001) doi:10.1006/abio.2001.5322, available online at http://www.idealibrary.com on
A New Reporter Gene System Suited for Cell-Free Protein Synthesis and High-Throughput Screening in Small Reaction Volumes Rene´ Hempel, Frank Wirsching, Andreas Schober,* and Andreas Schwienhorst 1 Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Goettingen, Germany; and *Institut fuer Physikalische Hochtechnologie (IPHT), Helmholtzweg 4, 07743 Jena, Germany
Received March 27, 2001; published online September 21, 2001
The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K ⴙ (and Rb ⴙ). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively. © 2001 Academic Press Key Words: in vitro translation; hirudin; luciferase reporter gene; cation concentration.
Studies of gene expression have been greatly simplified by the development of reporter gene constructs. Reporter genes in general code for proteins that exhibit special properties, e.g., enzymatic activities, easily distinguishable from the mixture of intra- or extracellular proteins. In recent years, reporter genes are of growing interest for high-throughput (HTS) 2 assays to monitor 1
To whom correspondence should be addressed. Fax: 49-551393805. E-mail: [email protected]. 2 Abbreviations used: AMC, 7-amino-4-methyl-coumarin; GFP, green fluorescent protein; IRES, internal ribosomal entry site; HCV, hepatitis C virus; HTS, high-throughput screening; uHTS, ultra high-throughput screening; MCA, 4-methyl-coumaryl-7-amide; Mhirudin, recombinant hirudin containing an additional methionine at the N-terminus; M1V hirudin, recombinant hirudin with valine at position 1 replaced by methionine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
gene activity (1–3). Unfortunately, many assays are not particularly well suited for HTS applications. Some systems such as CAT reporter systems (4, 5) are dependent on differential extraction steps and thus are quite time- and labor-intensive. Others such as the growth hormone reporter system (6) are expensive concerning the consumption of reagents. In addition, most reporter genes do not allow the sensitive detection of gene activity (6) and thus are not particularly well suited for assays in small reaction volumes, i.e., the wells of microtiter or even nanotiter plates (7). Furthermore, several reporter genes are only moderately suited for cell-free translation systems which become more and more important in the postgenomics era and drug design, e.g., the genome-wide screening (8), the development of antiviral agents (9 –11) and antitumor agents (12, 13), or the selection of novel proteins (14). The frequently used luciferase reporter system, for example, is highly dependent on concentrations of monovalent cations (15) and thus is not very robust against buffer variations. Herein we devise an alternative reporter system that combines a number of favorable properties. The new system is based on a variant of thrombin inhibitor hirudin from the leech Hirudo medicinalis. Previously, we have shown that recombinant hirudin could in some instances be detected as part of fusion proteins secreted into the periplasma of Escherichia coli (16). The variant of hirudin presented herein is recombinant hirudin extended by a single methionine at the Nterminus (M-hirudin). M-hirudin is less active than recombinant hirudin (17, 18). However, it still can be detected in nanomolar concentrations using a standard fluorimetric assay (16). 177
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The cell-free synthesis of M-hirudin in rabbit reticulocyte lysate is robust against large variations of concentrations in K ⫹ (and Rb ⫹) as compared to the luciferase system. In addition, the gene product can be detected with considerable sensitivity using a standard fluorimetric thrombin assay. We show that M-hirudin can be synthesized and assayed in small-volume nanotiter and microtiter plates. Altogether, M-hirudin seems to be well suited as a reporter gene in the highthroughput screening for drug candidates that specifically interact with specific mRNAs or the translation maschinery itself. This is particularly important for the discovery of antiviral drugs, e.g., antisense RNAs, that specifically interact with internal ribosomal entry sites in the 5⬘-UTR of viral RNAs such as hepatitis C virus RNA (9). MATERIALS AND METHODS
Materials Restriction enzymes and T4 DNA ligase were purchased from MBI Fermentas (St. Leon-Rot, Germany) and Vent polymerase from New England BioLabs (Schwalbach, Germany). All oligonucleotides and primers were obtained from Metabion GmbH (Martinsried, Germany). Plasmid preparations were performed using QIAprep (Qiagen, Hilden, Germany). Thrombin, AMC (7-amino-4-methyl-coumarin), and Tos-GlyPro-Arg-MCA were purchased from Sigma-Aldrich (Deisenhofen, Germany). Vector pSP6-Control DNA was supplied by Promega (Madison, WI). Construction of Vectors Different variants of hirudin genes were prepared by PCR using Vent polymerase and vector pCANTABHV1 (16) as a template. The variant containing an additional methionine codon at the N-terminus (M-hirudin) was amplified with primers MVV-FW (5⬘GCGAATTCGGGCCCAGATCTACCATGGTTGTTTACACTGC-3⬘) and HIR-RV (5⬘-GCGAATTCGAGCTCTTATGCGGCACGCGGTTC-3⬘). Hirudin gene variant M1V-hirudin containing a methionine codon replacing the N-terminal valine codon was amplified with primers MV-FW (5⬘-GCGAATTCGGGCCCAGATCTACCATGGTTTACACTGC-3⬘) and HIR-RV. PCR products were digested with ApaI and SacI and subcloned into the ApaI/SacI double-digested vector pGEM-11Zf(⫹) (Promega). Genes were cleaved with HindIII and SacI and ligated into the HindIII/SacI double-digested vector pSP64Poly(A) (Promega). In the final constructs, the genes contained an optimized translation start region (ACCATGG) and additional sequences at the C-terminus of the hirudin sequences coding for the
E-tag and a poly(A) tail. Vectors pSP64-M-HV1 and pSP64-M1V-HV1 were transformed into E. coli-strain XL1-Blue (Stratagene, La Jolla, CA). In Vitro Transcription Plasmid DNA was linearized with EcoRI and purified by phenol/chloroform extraction and ethanol precipitation. Runoff transcription was performed using the RiboMAX system (Promega) as described in the supplier’s manual. As a control, luciferase mRNA was synthesized from linearized vector Luciferase SP6Control DNA as supplied by Promega. By design, all transcripts contained a 3⬘ terminal (A) 30-tail and no capping at the 5⬘ terminus. Transcripts were purified by size exclusion HPLC as previously described (19). After ethanol precipitation and resuspension in nuclease-free water (Fluka, Deisenhofen, Germany) containing 0.4 U/l RNasin (Promega) the mRNA was stored at ⫺80°C. In Vitro Translation In vitro translation of runoff transcripts was accomplished using the Flexi Rabbit reticulocyte lysate system (Promega). Several batches were pooled to guarantee comparable conditions in parallel or successive reactions. In this way reproducible translation results could be achieved with standard deviations of ⱕ3% within 1–2 weeks after mRNA preparation (15). In contrast to the suppliers manual we used 5 l of lysate in a 10-l standard reaction. Smaller fractions of lysate lead to a significant decrease in translation yield. Standard translation conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, and additions of KCl or RbCl as indicated (15). mRNA was heated for 2 min at 95°C prior to addition to the translation cocktail. Translation was successfully performed for 3.5 h in final volumes ranging from 25 l down to 100 nl with appropriate downscaling of single components. Reactions were routinely carried out in 96-, 384-, and 1536-well-microtiter plates as well as specialized nanotiter plates (IPHT, Jena, Germany) manufactured from six segments of a silicon wafer (7, 20). Each segment comprises 900 reaction compartments with a maximum volume of 120 nl each. The (outer) dimensions of the nanotiter plates refer to international standard formats. During incubation, nanotiter plates were covered with standard lids. Standard Assay for Luciferase Activity Luciferase translation was directly monitored in a standard luciferase assay (Steady-Glo, Promega). Steady-Glo substrate was dissolved in Steady-Glo
REPORTER GENE SCREENING SYSTEM
buffer according to the instructions of the manufacturer. A standard reaction contained 5 l unpurified translation mixture and 45 l of dissolved Steady-Glo substrate. Luminometric measurements were carried out with the help of a robotic workstation (CyBiScreen-Machine, CyBio AG, Jena, Germany) including a Polarstar fluorescence reader (BMG, Offenburg, Germany). Assay data are mean values obtained from three independent translation reactions each of them measured at least twice. The amount of translated luciferase was detected by comparison with a standard of QuantiLum recombinant luciferase (Promega). The detection limit for luciferase was determined to be 81 fmol. Thrombin Inhibitory Assay Translation products, i.e., translation mixtures, were directly tested without prior purification. The inhibition of thrombin was quantified using the fluorogenic assay as described (16). Briefly, the fluorogenic substrate Tos-Gly-Pro-Arg-4-methyl-coumaryl-7-amide was hydrolyzed by thrombin releasing 7-amino-4methyl-coumarin (AMC). In this assay the detection limit for thrombin was determined to be 0.1 fmol. To test in vitro-translated products the assay was carried out by mixing 49 l assay buffer (0.05 M Tris, 0.1 M NaCl, 100 g/ml BSA, 0.1% PEG 8000, pH 7.6) with 10 l thrombin solution (10 ⫺5 U/l in assay buffer) and 0.3–1 l translation mixture. After incubation for 3 min at 30°C 140 l of a 30 M solution of fluorogenic substrate in assay buffer was added. Note, that it is important to keep the amount of translation mixtures per assay below 2 l since higher concentrations of mixtures even without hirudin variants turned out to yield significant thrombin inhibitory activity. The fluorescence of the enzymatically generated AMC was monitored for 30 min at a wavelength of 460 nm ( ex ⫽ 390 nm) as a function of time. The AMC signals were recorded against a blank with buffer and substrate but without the enzyme. Fluorescence intensities were calibrated with 7-amino-4-methylcoumarin. All assays were carried out with the help of a robotic workstation (CyBi™-Screen-Machine, CyBio AG, Jena, Germany) including a Polarstar fluorescence reader (BMG, Offenburg, Germany). Assay data are mean values obtained from three independent translation reactions each of them measured at least three times. As a negative control a translation mixture containing luciferase but no hirudin variants was used in the assay. In this paper data is presented as thrombin inhibitory activity ⫽ (thrombin activity without inhibitor) ⫺ (residual thrombin activity in the presence of inhibitor, i.e., translation mixture).
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Evaluation of Enzymatic Assays To evaluate the results obtained in the enzymatic assays, a large number of translation reactions were also tested for translation efficiency in the presence of radioactively labeled [ 35S]methionine. Relative incorporation of radioactivity into TCA precipitates as well as relative band counts after SDS–PAGE revealed the same relative translation efficiencies as compared to those obtained in the enzymatic assay (15). RESULTS AND DISCUSSION
Hirudin, a 65-amino-acid peptide from H. medicinalis, is currently the most potent thrombin inhibitor exhibiting K i constants in the subpicomolar range. Recombinant hirudin (r-hirudin) has been expressed in several systems, including Saccharomyces cerevisiae (21), E. coli (16, 22) and displayed on the surface of bacteriophage (16). It differs from the natural isolate only in the absence of tyrosine sulfation. The inhibition of thrombin by hirudin can be assayed most sensitively and highly specific, e.g., by using a fluorogenic substrate (16, 23). Altogether, the data suggest to use hirudin as an exceptionally small reporter protein. Previously, we could show, that recombinant hirudin is already well suited as a reporter molecule in vivo (16). As part of fusion proteins, however, it displays its thrombin inhibitory activity only at the N-terminus of the fusion protein. Extension or mutation of the Nterminal valine of hirudin is normally not tolerated (16 –18, 24, 25). Only replacement of the N-terminal valine by other hydrophobic residues resulted in minor changes in binding energy (18, 24). Since the original N-terminal valine codon in hirudin is rather inefficient as a start codon we designed genes of two hirudin variants, M-hirudin (containing an additional methionine codon at the N-terminus) and M1V-hirudin (containing an N-terminal methionine codon replacing the first valine codon). Both variants were cloned downstream of the SP6-promoter in plasmid pSP64Poly(A) to facilitate in vitro transcription/translation. Messenger RNA was obtained by runoff transcription using EcoRI-digested vectors. Synthesis of hirudin variants and luciferase was accomplished using the cell-free reticulocyte lysate system. Conditions for the cell-free translation of hirudin variants comprise final concentrations of 20 mM amino acids, 1.6 U/l RNasin, varying concentrations of RNA as indicated, and additions of 70 mM KCl. Aliquots of the translation mixtures were directly tested for activity using a standard fluorimetric assay. In agreement with previous findings, mutation of the N-terminal valine toward methionine leads to a variant practically inactive in the thrombin inhibitory assay (Fig. 1). However, an additional methionine codon
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Several applications of reporter genes, in particular in the postgenomics era require an extremely high sensitivity concerning the quantification of the gene product. We therefore determined the detection limit of M-hirudin from an in vitro translation reaction in a standard fluorimetric assay (Fig. 3). Using a microtiter plate fluorescence reader (BMG), we could still clearly detect the amount of M-hirudin (approx. 1 ng) from a
FIG. 1. Amidolytic thrombin inhibitory assay with (■) M-hirudin and (E) M1V hirudin. Both variants were expressed using the Flexi Rabbit reticulocyte lysate system (Promega, Madison, WI) with mRNA concentrations as indicated.
at the N-terminus does not abolish inhibition of thrombin. Thus, we chose M-hirudin as the reporter construct in subsequent experiments. In vitro transcript preparations, in particular those which have been precipated in the presence of salts, e.g., potassium salts, may contain significant and varying amounts of salt that could lead to suboptimal translation. To compare the hirudin reporter system with a standard luciferase system in regard to robustness against cations, a number of different concentrations of KCl and RbCl were tested for in vitro translation. Reaction conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, 0.4 mM Mg-acetate, and additions of 0 –140 mM KCl or RbCl, respectively, as indicated. Translation efficiency was monitored in subsequent enzymatic assays as described (Fig. 2). Experiments revealed, that the in vitro translation of M-hirudin was very efficient and remained at the same level throughout a concentration range of 20 –100 mM. In constrast, translation of the luciferase gene under the same conditions revealed a much more narrow and steep optimum at 80 mM (KCl) and 100 mM (RbCl), respectively. Since relative translation efficiencies were confirmed by radioactive incorporation experiments (data not shown), we tend to believe that M-hirudin translation is significantly more robust against variations of salt concentrations (at least of K ⫹ and Rb ⫹) as compared to luciferase translation. The addition of RbCl proved to be more stimulatory as compared to the potassium salt.
FIG. 2. Effect of monovalent cations on the in vitro translation efficacy of luciferase (open symbols) and M-hirudin (filled symbols). (A) KCl dependence; (B) dependence on different RbCl concentrations. Translation efficiency was monitored in subsequent luminometric (luciferase) or fluorimetric (hirudin) enzymatic assays.
REPORTER GENE SCREENING SYSTEM
FIG. 3. Determination of the detection limit of M-hirudin in a standard amidolytic thrombin inhibitory assay. M-hirudin was expressed using the Flexi Rabbit reticulocyte lysate system (Promega, WI). Different volumes of the translation mixture were analyzed in a standard fluorimetric thrombin assay.
300-nl standard in vitro translation reaction. This data already suggests that M-hirudin may very well be suited for high-throughput screening applications in small volumes. To confirm that the new reporter gene system is indeed appropriate for HTS assays, synthesis as well as fluorimetric assays of M-hirudin were carried out in macroscopic Eppendorf caps and nanotiter/microtiter plates (Fig. 4A) using a robotic workstation (CyBi-
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Screen-Machine, CyBio AG), including a Polarstar fluorescence reader (BMG). Final volumes of translation reactions were 10 l and 100 nl, respectively. Translation reaction conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, and additions of 70 mM KCl. Thrombin inhibitory activities per 1 l of either translation reactions (Eppendorf caps and nanotiter/microtiter plates) were 74.7 ⫾ 0.8 and 74.5 ⫾ 8.2%, respectively. Significantly larger standard deviations in the case of nanotiter plate-born translation reactions were presumably due to larger pipetting errors in the submicroliter range. In conclusion, thrombin inhibitory activities were comparable whatever sample carrier was used, indicating that also translation efficiencies were the same. As with any reporter technique that needs external reagents to produce a detectable signal, assays for Mhirudin are neccessarily multistep and somewhat more complicated as compared to, e.g., the GFP system. However, unlike GFP there are no (slow) kinetics of maturation, e.g., oxidation processes involved in signal generation (26) which also represent an additional source of experimental error. In summary, hirudins appear to be rather small reporter genes with attractive properties. Whereas unmodified recombinant hirudin is well suited as a reporter gene that is expressed in vivo, e.g., as part of periplasmic fusion proteins, M-hirudin appears to be a reporter protein that can be efficiently synthesized in vitro. In contrast to other reporter systems in vitro translation efficiencies are robust against variations in salt concentration. Furthermore, translation is effi-
FIG. 4. In vitro translation of M-hirudin in silicon wafers. (A) Sample carrier containing six segments of a silicon wafer (7, 20), each comprising 900 reaction compartments with a maximum volume of 120 nl each. (B) In vitro translation of M-hirudin in Eppendorf caps and silicon wafer segments using the Flexi Rabbit reticulocyte lysate system (Promega, Madison, WI). Translation efficiency was monitored in subsequent fluorimetric enzymatic assays. Inhibitory activity refers to the amount of 1 l of translation reaction in each case.
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cient in microtiter and nanotiter plates. Here, submicromolar concentrations of M-hirudin can be detected under standard assay conditions. Hence, M-hirudin should be the appropriate reporter gene for highthroughput applications, e.g., the discovery of novel inhibitors of the IRES-dependent translation of viruses such as HCV (9). ACKNOWLEDGMENTS The authors thank Dr. M. Gebinoga for many fruitful discussions and for reading the manuscript. Technical support from Cybio AG and BMG GmbH was greatly acknowledged. This work was supported in part by grant BioFuture 0311852, Grants 0311816 and 0311766 from the Bundesministerium fu¨r Forschung und Technologie and Grant Schw578/3 from the DFG. The authors thank G. Mayer and the PROMAG group and the TMWFK Thuringen for generous support.
REFERENCES 1. Groskreutz, D., and Schenborn, E. T. (1997) Methods Mol. Biol. 63, 11–30. 2. Silverman, L., Campbell, R., and Broach, J. R. (1998) Curr. Opin. Chem. Biol. 2, 397– 403. 3. Schenborn, E., and Groskreutz, D. (1999) Mol. Biotechnol. 13, 29 – 44. 4. Wynshaw-Boris, A., Short, J. M., and Hanson, R. W. (1986) BioTechniques 4, 104 –119. 5. Chen, C., and Okayama, H. (1987) Mol. Cell Biol. 7, 2745–2752. 6. Alam, J., and Cook, J. L. (1990) Anal. Biochem. 188, 245–254. 7. Ko¨hler, J. M., Schober, A., and Schwienhorst, A. (1994) Exp. Tech. Phys. 40, 35–56. 8. Tonjes, R. R., Czauderna, F., and Kurth, R. (1999) J. Virol. 73, 9187–9195. 9. Walker, M. A. (1999) Drug Discovery Today 4, 518 –529.
10. Gish, R. G. (1999) Can. J. Gastroenterol. 13, 57– 62. 11. Brown-Driver, V., Eto, T., Lesnik, E., Anderson, K. P., and Hanecak, R. C. (1999) Antisense Nucleic Acid Drug Dev. 9, 145–154. 12. Schlick, J., Dulieu, P., Desvoyes, B., Adami, P., J., R., and Jouvenot, M. (2000) FEBS Lett. 472, 241–246. 13. Beerheide, W., Bernard, H. U., Tan, Y. J., Ganesan, A., Rice, W. G., and Ting, A. E. (1999) J. Natl. Cancer Inst. 91, 1211–1220. 14. Doi, N., and Yanagawa, H. (1998) Cell. Mol. Life Sci. 54, 394 – 404. 15. Hempel, R., Schmidt-Brauns, J., Gebinoga, M., Wirsching, F., and Schwienhorst, A. (2001) Biochem. Biophys. Res. Commun. 283, 267–272. 16. Wirsching, F., Opitz, T., Dietrich, R., and Schwienhorst, A. (1997) Gene 204, 177–184. 17. Loison, G., Findeli, A., Bernard, S., Nguyen-Juilleret, M., Marquet, M., Riehl-Bellon, N., Carvallo, D., Guerra-Santos, L., Brown, S. W., Courtney, M., Roitsch, C., and Lemoine, Y. (1988) BioTechnology 6, 72–77. 18. Wallace, A., Dennis, S., Hofsteenge, J., and Stone, S. R. (1989) Biochemistry 28, 10,079 –10,084. 19. Pu¨tz, J., Wientges, J., Sissler, M., Giege´, R., Florentz, C., and Schwienhorst, A. (1997) Nucleic Acids Res. 25, 1862–1863. 20. Schober, A., Schwienhorst, A., Ko¨hler, J. M., Fuchs, M., Gu¨nther, R., and Thu¨rk, M. (1995) Microsyt. Technol. 1, 168 – 172. 21. Harvey, R. P., Degryse, E., Stefani, L., Schamber, F., Cazenave, J.-P., Courtney, M., Tolstoshev, P., and Lecocq, J.-P. (1986) Proc. Natl. Acad. Sci. USA 83, 1084 –1088. 22. Bergmann, C., Dodt, J., Ko¨hler, S., Fink, E., and Gassen, H. G. (1968) Biol. Chem. Hoppe-Seyler 367, 731–740. 23. Szewczuk, Z., Gibbs, B. F., Yue, S. Y., Purisima, E. O., and Konishi, Y. (1992) Biochemistry 31, 9132–9140. 24. Betz, A., Hofsteenge, J., and Stone, S. R. (1992) Biochemistry 31, 4557– 4562. 25. Fortkamp, E., Rieger, M., Heisterberg-Moutses, G., Schweitzer, S., and Sommer, R. (1986) DNA 5, 511–517. 26. Reid, B. G., and Flynn, G. C. (1997) Biochemistry 36, 6786 – 6791.
A New Reporter Gene System Suited for Cell-Free Protein Synthesis and High-Throughput Screening in Small Reaction Volumes Rene´ Hempel, Frank Wirsching, Andreas Schober,* and Andreas Schwienhorst 1 Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Goettingen, Germany; and *Institut fuer Physikalische Hochtechnologie (IPHT), Helmholtzweg 4, 07743 Jena, Germany
Received March 27, 2001; published online September 21, 2001
The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K ⴙ (and Rb ⴙ). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively. © 2001 Academic Press Key Words: in vitro translation; hirudin; luciferase reporter gene; cation concentration.
Studies of gene expression have been greatly simplified by the development of reporter gene constructs. Reporter genes in general code for proteins that exhibit special properties, e.g., enzymatic activities, easily distinguishable from the mixture of intra- or extracellular proteins. In recent years, reporter genes are of growing interest for high-throughput (HTS) 2 assays to monitor 1
To whom correspondence should be addressed. Fax: 49-551393805. E-mail: [email protected]. 2 Abbreviations used: AMC, 7-amino-4-methyl-coumarin; GFP, green fluorescent protein; IRES, internal ribosomal entry site; HCV, hepatitis C virus; HTS, high-throughput screening; uHTS, ultra high-throughput screening; MCA, 4-methyl-coumaryl-7-amide; Mhirudin, recombinant hirudin containing an additional methionine at the N-terminus; M1V hirudin, recombinant hirudin with valine at position 1 replaced by methionine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
gene activity (1–3). Unfortunately, many assays are not particularly well suited for HTS applications. Some systems such as CAT reporter systems (4, 5) are dependent on differential extraction steps and thus are quite time- and labor-intensive. Others such as the growth hormone reporter system (6) are expensive concerning the consumption of reagents. In addition, most reporter genes do not allow the sensitive detection of gene activity (6) and thus are not particularly well suited for assays in small reaction volumes, i.e., the wells of microtiter or even nanotiter plates (7). Furthermore, several reporter genes are only moderately suited for cell-free translation systems which become more and more important in the postgenomics era and drug design, e.g., the genome-wide screening (8), the development of antiviral agents (9 –11) and antitumor agents (12, 13), or the selection of novel proteins (14). The frequently used luciferase reporter system, for example, is highly dependent on concentrations of monovalent cations (15) and thus is not very robust against buffer variations. Herein we devise an alternative reporter system that combines a number of favorable properties. The new system is based on a variant of thrombin inhibitor hirudin from the leech Hirudo medicinalis. Previously, we have shown that recombinant hirudin could in some instances be detected as part of fusion proteins secreted into the periplasma of Escherichia coli (16). The variant of hirudin presented herein is recombinant hirudin extended by a single methionine at the Nterminus (M-hirudin). M-hirudin is less active than recombinant hirudin (17, 18). However, it still can be detected in nanomolar concentrations using a standard fluorimetric assay (16). 177
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The cell-free synthesis of M-hirudin in rabbit reticulocyte lysate is robust against large variations of concentrations in K ⫹ (and Rb ⫹) as compared to the luciferase system. In addition, the gene product can be detected with considerable sensitivity using a standard fluorimetric thrombin assay. We show that M-hirudin can be synthesized and assayed in small-volume nanotiter and microtiter plates. Altogether, M-hirudin seems to be well suited as a reporter gene in the highthroughput screening for drug candidates that specifically interact with specific mRNAs or the translation maschinery itself. This is particularly important for the discovery of antiviral drugs, e.g., antisense RNAs, that specifically interact with internal ribosomal entry sites in the 5⬘-UTR of viral RNAs such as hepatitis C virus RNA (9). MATERIALS AND METHODS
Materials Restriction enzymes and T4 DNA ligase were purchased from MBI Fermentas (St. Leon-Rot, Germany) and Vent polymerase from New England BioLabs (Schwalbach, Germany). All oligonucleotides and primers were obtained from Metabion GmbH (Martinsried, Germany). Plasmid preparations were performed using QIAprep (Qiagen, Hilden, Germany). Thrombin, AMC (7-amino-4-methyl-coumarin), and Tos-GlyPro-Arg-MCA were purchased from Sigma-Aldrich (Deisenhofen, Germany). Vector pSP6-Control DNA was supplied by Promega (Madison, WI). Construction of Vectors Different variants of hirudin genes were prepared by PCR using Vent polymerase and vector pCANTABHV1 (16) as a template. The variant containing an additional methionine codon at the N-terminus (M-hirudin) was amplified with primers MVV-FW (5⬘GCGAATTCGGGCCCAGATCTACCATGGTTGTTTACACTGC-3⬘) and HIR-RV (5⬘-GCGAATTCGAGCTCTTATGCGGCACGCGGTTC-3⬘). Hirudin gene variant M1V-hirudin containing a methionine codon replacing the N-terminal valine codon was amplified with primers MV-FW (5⬘-GCGAATTCGGGCCCAGATCTACCATGGTTTACACTGC-3⬘) and HIR-RV. PCR products were digested with ApaI and SacI and subcloned into the ApaI/SacI double-digested vector pGEM-11Zf(⫹) (Promega). Genes were cleaved with HindIII and SacI and ligated into the HindIII/SacI double-digested vector pSP64Poly(A) (Promega). In the final constructs, the genes contained an optimized translation start region (ACCATGG) and additional sequences at the C-terminus of the hirudin sequences coding for the
E-tag and a poly(A) tail. Vectors pSP64-M-HV1 and pSP64-M1V-HV1 were transformed into E. coli-strain XL1-Blue (Stratagene, La Jolla, CA). In Vitro Transcription Plasmid DNA was linearized with EcoRI and purified by phenol/chloroform extraction and ethanol precipitation. Runoff transcription was performed using the RiboMAX system (Promega) as described in the supplier’s manual. As a control, luciferase mRNA was synthesized from linearized vector Luciferase SP6Control DNA as supplied by Promega. By design, all transcripts contained a 3⬘ terminal (A) 30-tail and no capping at the 5⬘ terminus. Transcripts were purified by size exclusion HPLC as previously described (19). After ethanol precipitation and resuspension in nuclease-free water (Fluka, Deisenhofen, Germany) containing 0.4 U/l RNasin (Promega) the mRNA was stored at ⫺80°C. In Vitro Translation In vitro translation of runoff transcripts was accomplished using the Flexi Rabbit reticulocyte lysate system (Promega). Several batches were pooled to guarantee comparable conditions in parallel or successive reactions. In this way reproducible translation results could be achieved with standard deviations of ⱕ3% within 1–2 weeks after mRNA preparation (15). In contrast to the suppliers manual we used 5 l of lysate in a 10-l standard reaction. Smaller fractions of lysate lead to a significant decrease in translation yield. Standard translation conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, and additions of KCl or RbCl as indicated (15). mRNA was heated for 2 min at 95°C prior to addition to the translation cocktail. Translation was successfully performed for 3.5 h in final volumes ranging from 25 l down to 100 nl with appropriate downscaling of single components. Reactions were routinely carried out in 96-, 384-, and 1536-well-microtiter plates as well as specialized nanotiter plates (IPHT, Jena, Germany) manufactured from six segments of a silicon wafer (7, 20). Each segment comprises 900 reaction compartments with a maximum volume of 120 nl each. The (outer) dimensions of the nanotiter plates refer to international standard formats. During incubation, nanotiter plates were covered with standard lids. Standard Assay for Luciferase Activity Luciferase translation was directly monitored in a standard luciferase assay (Steady-Glo, Promega). Steady-Glo substrate was dissolved in Steady-Glo
REPORTER GENE SCREENING SYSTEM
buffer according to the instructions of the manufacturer. A standard reaction contained 5 l unpurified translation mixture and 45 l of dissolved Steady-Glo substrate. Luminometric measurements were carried out with the help of a robotic workstation (CyBiScreen-Machine, CyBio AG, Jena, Germany) including a Polarstar fluorescence reader (BMG, Offenburg, Germany). Assay data are mean values obtained from three independent translation reactions each of them measured at least twice. The amount of translated luciferase was detected by comparison with a standard of QuantiLum recombinant luciferase (Promega). The detection limit for luciferase was determined to be 81 fmol. Thrombin Inhibitory Assay Translation products, i.e., translation mixtures, were directly tested without prior purification. The inhibition of thrombin was quantified using the fluorogenic assay as described (16). Briefly, the fluorogenic substrate Tos-Gly-Pro-Arg-4-methyl-coumaryl-7-amide was hydrolyzed by thrombin releasing 7-amino-4methyl-coumarin (AMC). In this assay the detection limit for thrombin was determined to be 0.1 fmol. To test in vitro-translated products the assay was carried out by mixing 49 l assay buffer (0.05 M Tris, 0.1 M NaCl, 100 g/ml BSA, 0.1% PEG 8000, pH 7.6) with 10 l thrombin solution (10 ⫺5 U/l in assay buffer) and 0.3–1 l translation mixture. After incubation for 3 min at 30°C 140 l of a 30 M solution of fluorogenic substrate in assay buffer was added. Note, that it is important to keep the amount of translation mixtures per assay below 2 l since higher concentrations of mixtures even without hirudin variants turned out to yield significant thrombin inhibitory activity. The fluorescence of the enzymatically generated AMC was monitored for 30 min at a wavelength of 460 nm ( ex ⫽ 390 nm) as a function of time. The AMC signals were recorded against a blank with buffer and substrate but without the enzyme. Fluorescence intensities were calibrated with 7-amino-4-methylcoumarin. All assays were carried out with the help of a robotic workstation (CyBi™-Screen-Machine, CyBio AG, Jena, Germany) including a Polarstar fluorescence reader (BMG, Offenburg, Germany). Assay data are mean values obtained from three independent translation reactions each of them measured at least three times. As a negative control a translation mixture containing luciferase but no hirudin variants was used in the assay. In this paper data is presented as thrombin inhibitory activity ⫽ (thrombin activity without inhibitor) ⫺ (residual thrombin activity in the presence of inhibitor, i.e., translation mixture).
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Evaluation of Enzymatic Assays To evaluate the results obtained in the enzymatic assays, a large number of translation reactions were also tested for translation efficiency in the presence of radioactively labeled [ 35S]methionine. Relative incorporation of radioactivity into TCA precipitates as well as relative band counts after SDS–PAGE revealed the same relative translation efficiencies as compared to those obtained in the enzymatic assay (15). RESULTS AND DISCUSSION
Hirudin, a 65-amino-acid peptide from H. medicinalis, is currently the most potent thrombin inhibitor exhibiting K i constants in the subpicomolar range. Recombinant hirudin (r-hirudin) has been expressed in several systems, including Saccharomyces cerevisiae (21), E. coli (16, 22) and displayed on the surface of bacteriophage (16). It differs from the natural isolate only in the absence of tyrosine sulfation. The inhibition of thrombin by hirudin can be assayed most sensitively and highly specific, e.g., by using a fluorogenic substrate (16, 23). Altogether, the data suggest to use hirudin as an exceptionally small reporter protein. Previously, we could show, that recombinant hirudin is already well suited as a reporter molecule in vivo (16). As part of fusion proteins, however, it displays its thrombin inhibitory activity only at the N-terminus of the fusion protein. Extension or mutation of the Nterminal valine of hirudin is normally not tolerated (16 –18, 24, 25). Only replacement of the N-terminal valine by other hydrophobic residues resulted in minor changes in binding energy (18, 24). Since the original N-terminal valine codon in hirudin is rather inefficient as a start codon we designed genes of two hirudin variants, M-hirudin (containing an additional methionine codon at the N-terminus) and M1V-hirudin (containing an N-terminal methionine codon replacing the first valine codon). Both variants were cloned downstream of the SP6-promoter in plasmid pSP64Poly(A) to facilitate in vitro transcription/translation. Messenger RNA was obtained by runoff transcription using EcoRI-digested vectors. Synthesis of hirudin variants and luciferase was accomplished using the cell-free reticulocyte lysate system. Conditions for the cell-free translation of hirudin variants comprise final concentrations of 20 mM amino acids, 1.6 U/l RNasin, varying concentrations of RNA as indicated, and additions of 70 mM KCl. Aliquots of the translation mixtures were directly tested for activity using a standard fluorimetric assay. In agreement with previous findings, mutation of the N-terminal valine toward methionine leads to a variant practically inactive in the thrombin inhibitory assay (Fig. 1). However, an additional methionine codon
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Several applications of reporter genes, in particular in the postgenomics era require an extremely high sensitivity concerning the quantification of the gene product. We therefore determined the detection limit of M-hirudin from an in vitro translation reaction in a standard fluorimetric assay (Fig. 3). Using a microtiter plate fluorescence reader (BMG), we could still clearly detect the amount of M-hirudin (approx. 1 ng) from a
FIG. 1. Amidolytic thrombin inhibitory assay with (■) M-hirudin and (E) M1V hirudin. Both variants were expressed using the Flexi Rabbit reticulocyte lysate system (Promega, Madison, WI) with mRNA concentrations as indicated.
at the N-terminus does not abolish inhibition of thrombin. Thus, we chose M-hirudin as the reporter construct in subsequent experiments. In vitro transcript preparations, in particular those which have been precipated in the presence of salts, e.g., potassium salts, may contain significant and varying amounts of salt that could lead to suboptimal translation. To compare the hirudin reporter system with a standard luciferase system in regard to robustness against cations, a number of different concentrations of KCl and RbCl were tested for in vitro translation. Reaction conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, 0.4 mM Mg-acetate, and additions of 0 –140 mM KCl or RbCl, respectively, as indicated. Translation efficiency was monitored in subsequent enzymatic assays as described (Fig. 2). Experiments revealed, that the in vitro translation of M-hirudin was very efficient and remained at the same level throughout a concentration range of 20 –100 mM. In constrast, translation of the luciferase gene under the same conditions revealed a much more narrow and steep optimum at 80 mM (KCl) and 100 mM (RbCl), respectively. Since relative translation efficiencies were confirmed by radioactive incorporation experiments (data not shown), we tend to believe that M-hirudin translation is significantly more robust against variations of salt concentrations (at least of K ⫹ and Rb ⫹) as compared to luciferase translation. The addition of RbCl proved to be more stimulatory as compared to the potassium salt.
FIG. 2. Effect of monovalent cations on the in vitro translation efficacy of luciferase (open symbols) and M-hirudin (filled symbols). (A) KCl dependence; (B) dependence on different RbCl concentrations. Translation efficiency was monitored in subsequent luminometric (luciferase) or fluorimetric (hirudin) enzymatic assays.
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FIG. 3. Determination of the detection limit of M-hirudin in a standard amidolytic thrombin inhibitory assay. M-hirudin was expressed using the Flexi Rabbit reticulocyte lysate system (Promega, WI). Different volumes of the translation mixture were analyzed in a standard fluorimetric thrombin assay.
300-nl standard in vitro translation reaction. This data already suggests that M-hirudin may very well be suited for high-throughput screening applications in small volumes. To confirm that the new reporter gene system is indeed appropriate for HTS assays, synthesis as well as fluorimetric assays of M-hirudin were carried out in macroscopic Eppendorf caps and nanotiter/microtiter plates (Fig. 4A) using a robotic workstation (CyBi-
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Screen-Machine, CyBio AG), including a Polarstar fluorescence reader (BMG). Final volumes of translation reactions were 10 l and 100 nl, respectively. Translation reaction conditions comprise final concentrations of 80 ng/l mRNA, 20 mM amino acids, 1.6 U/l RNasin, and additions of 70 mM KCl. Thrombin inhibitory activities per 1 l of either translation reactions (Eppendorf caps and nanotiter/microtiter plates) were 74.7 ⫾ 0.8 and 74.5 ⫾ 8.2%, respectively. Significantly larger standard deviations in the case of nanotiter plate-born translation reactions were presumably due to larger pipetting errors in the submicroliter range. In conclusion, thrombin inhibitory activities were comparable whatever sample carrier was used, indicating that also translation efficiencies were the same. As with any reporter technique that needs external reagents to produce a detectable signal, assays for Mhirudin are neccessarily multistep and somewhat more complicated as compared to, e.g., the GFP system. However, unlike GFP there are no (slow) kinetics of maturation, e.g., oxidation processes involved in signal generation (26) which also represent an additional source of experimental error. In summary, hirudins appear to be rather small reporter genes with attractive properties. Whereas unmodified recombinant hirudin is well suited as a reporter gene that is expressed in vivo, e.g., as part of periplasmic fusion proteins, M-hirudin appears to be a reporter protein that can be efficiently synthesized in vitro. In contrast to other reporter systems in vitro translation efficiencies are robust against variations in salt concentration. Furthermore, translation is effi-
FIG. 4. In vitro translation of M-hirudin in silicon wafers. (A) Sample carrier containing six segments of a silicon wafer (7, 20), each comprising 900 reaction compartments with a maximum volume of 120 nl each. (B) In vitro translation of M-hirudin in Eppendorf caps and silicon wafer segments using the Flexi Rabbit reticulocyte lysate system (Promega, Madison, WI). Translation efficiency was monitored in subsequent fluorimetric enzymatic assays. Inhibitory activity refers to the amount of 1 l of translation reaction in each case.
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cient in microtiter and nanotiter plates. Here, submicromolar concentrations of M-hirudin can be detected under standard assay conditions. Hence, M-hirudin should be the appropriate reporter gene for highthroughput applications, e.g., the discovery of novel inhibitors of the IRES-dependent translation of viruses such as HCV (9). ACKNOWLEDGMENTS The authors thank Dr. M. Gebinoga for many fruitful discussions and for reading the manuscript. Technical support from Cybio AG and BMG GmbH was greatly acknowledged. This work was supported in part by grant BioFuture 0311852, Grants 0311816 and 0311766 from the Bundesministerium fu¨r Forschung und Technologie and Grant Schw578/3 from the DFG. The authors thank G. Mayer and the PROMAG group and the TMWFK Thuringen for generous support.
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