AbstractA simple, cost-efficient method for purification of E. coli-derived
recombinant Taq DNA polymerase isolated from the thermophilic bac-terium Thermus aquaticus is described. Recombinant Taq DNA poly-merase was purified using an aqueous two-phase extraction method following a primary treatment of heat denaturation. The extraction method selectively enriched Taq DNA polymerase in the lower salt phase with complete recovery and high specific activity. The purified Taq DNA polymerase protein displayed nearly eightfold purification with ~117 % activity recovery with an insignificant amount of host DNA. This process was found to be rapid and scalable in comparison to the conventional ammonium sulfate precipitation method.
Introduction
DNA polymerase from Thermus aquaticus (Taq DNA poly-merase) is a DNA-dependent deoxynucleotidyltransfer-ase that was first isolated and purified by Chein et al.1 Purification of E. coli-derived recombinant Taq DNA
polymerase was later achieved by Lawyer et al.2 Since its introduc-tion, thermostable Taq DNA polymerase has found applications in molecular biology for amplification of DNA fragments by polymerase chain reaction (PCR) and in DNA sequencing protocols. To meet substantial demand, enzyme manufacturers and research laboratories would benefit from optimized, robust, and cost-efficient protocols for attaining high recovery of the purified enzyme.
The traditional methods for Taq pol purification include heat dena-turation accompanied by ammonium sulfate precipitation,3 affinity chromatography using nucleotide-mimetic ligands,4 and polyethyl-enimine precipitation followed by ion-exchange chromatography.5 Amongst these, the most common is the ammonium sulfate precipita-tion method. One problem associated with each of these procedures is the necessity of multiple purification steps, which renders the processes cost-intensive. To address this, we have attempted a simple aqueous phase system for purification of recombinant Taq DNA poly-merase expressed in E. coli.
An aqueous two-phase system is a biphasic liquid-liquid system obtained by mixing an aqueous solution of two polymers or by mix-ing a polymer and salt.6-8 Of various two-phase systems known, the polyethylene glycol (PEG)-salt system is more attractive, given the low cost of the salts and a higher selectivity of protein partitioning that results in enriched product with high yields.
The commonly used aqueous extraction method involves the mix-ture of two aqueous solutions which, when combined at a specific concentration, form two distinct phases according to the pH and ionic strength of the two components employed.9 This extraction method is frequently used for protein purification, since the proteins in the mix-
An alternate method for purification of recombinant Taq DNA polymerase using an aqueous two-phase system
ture are separated into one of three fractions, namely, the polymer phase, salt phase, or interphase, based on pI value, hydrophobicity, molecular weight, and ionic strength.10 Among the various PEG-salt systems reported, the PEG 4000–sodium sulfate combination is favored for protein purification, as its phase composition remains stable irrespective of protein concentration.11
In this paper, we illustrate to our knowledge the first use of an aqueous two-phase system (ATPS) using the PEG 4000-sodium sulfate combination for purification of recombinant Taq DNA poly-merase that avoids the use of costly matrices for purification and is comfortably scalable, with higher specific activity versus other prevalent purification methods.
Materials & methodsCONSTRUCTION OF PET21A-TAQ DNA POL I & EXPRESSION STUDIES
Taq DNA polymerase gene (Taq DNA Pol I) isolated from T. aquati-cus YT-1 strain (ATCC 25104) was cloned under the T7 promoter system and expressed in E. coli Rosetta-gami (DE3) pLysS cells (Novagen/EMD Biosciences, Gibbstown, New Jersey, USA). The cells were induced with 0.05 mM IPTG at 20˚C for 16 h.
PURIFICATION OF RECOMBINANT TAQ DNA POLYMERASEThe harvested cells (~2 g wet weight) containing the expressed
protein were resuspended in 30 mL of 10mM Tris-CL, pH 8.0, followed by cell disruption using a Nano Deebee Benchtop High Pressure Homogenizer (Bee International, South Easton, Massachusetts, USA) vacuum cell disruptor at 25 000 psi for 3 min. The disrupted cells were centrifuged and the supernatant incubated at 70˚C for 1 h. The treated sample was recentrifuged, and the clari-fied supernatant was distributed in two equal portions for purifica-tion studies by ATPS and by the ammonium sulfate precipitation method, independently.
AQUEOUS TWO-PHASE SYSTEM The clarified heat-treated lysate was subjected to the two-phase
system using PEG 4000 and sodium sulfate. The optimal conditions for this extraction method were 10% w/w PEG 4000 and 8% w/w sodium sulfate, with 82% w/w lysate at 25˚C. Separation of the two phases was expedited by centrifugation at 7 500 rpm for 10 min. The lower salt phase was dialyzed against 100 volumes of 10 mM Tris-CL, pH 8.0 at 4–8˚C for 16 h, and SDS-PAGE analysis was carried out for all samples.
AMMONIUM SULFATE PRECIPITATIONIn order to compare the ATPS method with the well-established
and widely used ammonium sulfate precipitation protocol, solid ammonium sulfate was added to the heat-treated supernatant at 30% saturation (176 g/L) and the mixture allowed to stand on ice for 30 min with stirring. The resulting precipitate was resuspended in 10 mM Tris-CL, pH 8.0, and dialyzed overnight against 100 volumes of 10 mM Tris pH.8.0 at 4–8˚C.
PROTEIN ESTIMATION & ACTIVITY ASSAYTotal protein from each step was estimated using the BCA pro-
tein assay kit (Thermo Scientific, Waltham, Massachusetts, USA) with BSA as the standard.12 The amount of Taq pol from each step was quantified by densitometric scanning using Quantity One software (Bio-Rad Laboratories, Hercules, California, USA). Taq pol preparations were assessed following the method of Grimm and Arbuthnot.13 In the current study, the staphylokinase (sak) gene (411 bp) was taken as a template for PCR using samples from each step of the purification as the source of Taq DNA polymerase, using the primer set as described earlier.14 Known units of commercial Taq DNA polymerase (Bangalore Genei/Merck; Bangalore, India) were used for a positive control, and relative intensities of the PCR products obtained were assessed by densitometry scanning, with activity units determined accordingly.
QUANTITATIVE REAL-TIME PCR HOST CELL DNA ESTIMATION
Total DNA was isolated from samples at each step of the purifi-cation following the ATPS method, using a DNeasy DNA isolation kit (Qiagen, Venlo, The Netherlands). Quantitative PCR was carried out in a StepOne™ real-time PCR machine (Applied Biosystems, Foster City, California, USA) to amplify host DNA using 16S rDNA-specific primers (Forward: 5´ GTGTAGCGGTGAAATGC 3; Reverse: 5´TGAGTTTTAACCTTGCGG3´) to determine the presence of host DNA in each phase.
TAQ DNA POLYMERASE SPECIFICITY & EFFICIENCY ASSAY
Quantitative real-time PCR (qPCR) was carried out using SYBR green chemistry (Applied Biosystems) in a StepOne real-time PCR machine (Applied Biosystems) to amplify the bla gene using gene-specific primers as reported elsewhere.15 Equal amounts of purified commercial Taq DNA polymerase preparation (Bangalore Genei) and in-house ATPS-purified Taq pol were used in order to determine the relative cycle threshold (CT) value or magnitude of the signal (ΔRn). The reaction was stopped after 25 cycles; the CT values indicated the relative activity of Taq pol of the two different preparations.
Results EXPRESSION OF TAQ DNA POLYMERASE
Rosetta-gami cells expressing tRNAs for six rare codons in the Taq DNA Pol I gene were used to express recombinant Taq DNA poly-merase of ~91 kDa. The majority of the Taq DNA polymerase protein expressed from the pET21a-TaqDNA Pol I construct was limited to the soluble fraction of the cell (data not shown); this was used as the starting material for purification studies.
PURIFICATION OF RECOMBINANT TAQ DNA POLYMERASE USING AQUEOUS TWO-PHASE SYSTEM
The aqueous two-phase system using a PEG-salt mixture was employed as described by Kepka et al.16 Crude cell lysate was sub-
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jected to heat at 70˚C for 1 h to precipitate the heat-labile proteins and to inactivate heat-labile host-derived proteases. This clarified supernatant was used as the starting material for ATPS purification, as depicted in the flow chart in Figure 1. It was observed that a highly pure Taq DNA polymerase protein was restricted to the salt phase (Figure 2A, Lane 4), leaving the majority of the host-contaminating proteins partitioned in the interphase (Figure 2A, Lane 5). This result was substantiated by peak profile analysis (Figure 2B) of the puri-fied Taq pol preparation using Bio-Rad (Hercules, California, USA) Quantity One analysis software.
To determine the amount of host DNA in various phases of ATPS, we carried out qRT-PCR with the genomic DNA from each phase of the two-phase system using primers specific to 16S rDNA. Interestingly, during this process of Taq pol purification, all the host DNAs (which could be interfering with PCR reactions carried out by the Taq polymerase protein) were restricted to the interphase and PEG
phase, as evident from the qRT PCR results (Figure 3). This suggests further advantages to the protocol described herein. Furthermore, the residual PEG (< 1.8%),11 which could not be removed by dialysis, did not appear to interfere with the PCR reaction.
AMMONIUM SULFATE PURIFICATION PROFILE OF TAQ DNA POLYMERASE
The heat-treated centrifuged supernatant was subjected to 30% ammonium sulfate saturation by addition of 176 g of solid ammo-nium sulfate per liter of solution, with constant stirring, in an ice bath. The SDS-PAGE profile showed that the majority of the Taq pol protein precipitated at this concentration of ammonium sulfate, although along with significant amounts of host proteins (Figure 4A, Lane 4). The purity of the Taq DNA polymerase protein was found to be nearly 35% (Figure 4B), in comparison to that achieved by the ATPS method (95%).
Figure 1. Flow chart showing parallel purification steps of recombinant Taq DNA polymerase using the aqueous two-phase and ammonium sul-fate precipitation methods. Inset illustrates the PEG-sodium sulfate two-phase system.
Whole cell lysate
Supernatant
Pelletcontaining heat-denatured
thermolabile impurities
Heated 70°C, 1 h,centrifuged at 13 000 rpm for 5 min
Resuspended in 10 mM Tris pH 8.0 and dialyzed overnight against 100 volumes of 10 mM Tris ph 8.0 in cold
Interphase(containing impurities)
PEG phase(containing impurities)
Supernatant(containing impurities)
Centrifuged at13 000 rpmfor 15 min
PEG phase
Interphase
Salt phase
PROTEIN YIELD ESTIMATION & ACTIVITY ASSAY
Protein yield in the initial and the heat-treated super-natants were found to be similar (~3.7 mg mL-1 and 3.6 mg mL-1, respectively). While the ammonium sulfate precipitated sample showed nearly 1.1 mg mL-1, the upper PEG layer contained ~11 mg protein, and the lower salt layer ~5.6 mg pro-tein, with the contaminating proteins (~18 mg) largely located within the inter-phase. These samples were analyzed for Taq pol activity by PCR (Figure 5A). Rresults show that DNA polymerizing activity is restricted solely to the salt phase sample (Lane 5). In the ammonium sulfate precipitation method, too, Taq pol activity was restrict-ed in the precipitate fraction,
as anticipated (data not shown). A specific activity of 637 and a purification factor of 7.9 were obtained for ATPS-purified Taq pol, while a specific activity of 207 and a purification factor of 2.6 were observed with for the ammonium sulfate-purified Taq pol (Table 1).
In order to validate the above assay results, the PCR reaction was also carried out with different dilutions of commercial Taq DNA polymerase (Bangalore Genei) and in-house ATPS-purified Taq DNA polymerase, with the reaction terminated after 18 cycles without extension step. Results indicated that the amount of PCR product formed increased with increasing concentration of Taq DNA poly-merase preparations (data not shown). This confirmed that the PCR reaction was indeed specific for Taq DNA polymerase.
When qPCR was carried out using the same concentration of com-mercial and in-house ATPS-purified Taq DNA polymerase prepara-tions, with the same template concentration, the CT value of the reac-tion with the ATPS-derived Taq DNA polymerase was 4.3, while that of the commercial Taq DNA polymerase was 13.8. Early and rapid amplification of the bla gene (Figure 5B), observed with the ATPS-purified Taq DNA polymerase, is indicative of a higher activity of the in-house purified Taq polymerase.
DiscussionSoluble heat-stable Taq DNA polymerase simplifies downstream
operations for lab- and large-scale manufacturing protocols. The common purification protocol employed in industry includes a salt
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Figure 2. Purification profile of Taq DNA polymerase using the aqueous two-phase system (ATPS). (A) SDS-PAGE followed by Coomassie Blue staining was carried out with equal-volume samples from different Taq polymerase purification stages of the ATPS. Lane 1, protein molecular weight marker (97-14 kDa); Lane 2, crude cell lysate containing expressed Taq; Lane 3, heat-treated (70˚C, 1 h) clarified lysate; Lane 4: dialyzed lower-salt phase; Lane 5, interphase sample. (B) Graphical representation of the purification profile. Relative intensity of each band was plotted against Rf value of the same. (1) Starting material, heat-treated cleared lysate;(2) Interphase containing impurities; (3) Salt phase containing Taq pol after dialysis. Arrow indicates peak of Taq DNA polymerase.
Figure 3. Quantitative RT-PCR for 16S rRNA showing the distribu-tion of host DNA in different phases of ATPS. Total DNA was isolated from each phase of the ATPS system, and qPCR was carried out in duplicate with 16S rRNA-specific primers. C T values were calculated by plotting number of cycles against ΔRn (baseline-corrected normal-ized reporter).
1 2 3 4 5kDa
97
66
43
29
A B 1
2
3
Rf
Rela
tive
inte
nsity
Rela
tive
inte
nsity
Rela
tive
inte
nsity
Amplification plot
PEG = Polyethylene glycol3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
-0.25
ΔR n
Cycle2 4 6 8 10 12 14 16 18 20 22 24
PEG phase
Salt phase
Interphase
precipitation step using ammo-nium sulfate.3 In the present puri-fication protocol, we have used a distinct approach to obtain Taq DNA polymerase with high specific activity. We have intro-duced an initial high-temperature denaturation step (70˚C) to elimi-nate and inactivate the majority of heat-labile host proteins. To our surprise, not many proteins were precipitated by this step, with only a negligible differ-ence observed between the SDS-PAGE profiles of the untreated and heat-treated samples. Our observations agreed with those of Kwon et al,17 who proposed that proteins remain soluble even in the unfolded state and refold back to their native structures when the temperature is returned to normal; hence no difference in SDS-PAGE profiles is seen for heat-treated and untreated sam-ples. Although protein precipita-tion by heat treatment is known to work optimally between 80˚C and 90˚C, a temperature of 70˚C was applied for the initial heat denaturation step for Taq DNA polymerase purification (since higher temperatures are known to reduce the amount of active DNA polymerase available for amplification18).
We did not observe a sig-nificant reduction of the protein concentration upon heat treat-ment; it could be contemplated that some of the E. coli prote-ases that would have become inactivated in the process could account for the higher total activity recovery of Taq pol in the heat-treated sample in com-parison to the starting material. Moreover, the unfolding of the contaminating proteins by heat would have contributed to the specific separation of Taq poly-merase in the salt layer.
Figure 4. Purification profile of Taq DNA polymerase using ammonium sulfate. (A) SDS-PAGE followed by Coomassie Blue staining was carried out with equal-volume samples of different purification stages of Taq DNA polymerase. Lane 1, protein molecular weight marker (97–14 kDa); Lane 2, crude cell lysate contain-ing expressed Taq; Lane 3, heat-treated (70˚C, 1 h) clarified lysate; Lane 4, purified Taq DNA polymerase using ammonium sulfate precipitation after dialysis. (B) Graphical representation of the purification profile. Relative intensity of each band was plotted against Rf value of the same. (1) Starting material, heat-treated cleared lysate; (2) 0% – 30% ammonium sulfate precipitate after dialysis. Arrow indicates Taq DNA poly-merase.
Figure 5. Quantitation of Taq DNA polymerase activity. (A) PCR amplification of the staphylokinase gene using different amounts of Taq pol samples from different purification stages. One hundred nanograms of synthetic gene corresponding to the staphylokinase gene was amplified using specific primers and Taq pol samples from different phases. Lane 1, l-DNA EcoRI/Hind III molecular weight marker; Lane 2, commercial Taq DNA polymerase; Lane 3, 5 µL heat-treated cell lysate; Lane 4, 5 µL of PEG layer (upper layer); Lane 5, 5 µL salt layer (lower layer); Lane 6, negative control. (B) Amplification plot; qPCR was carried out for the bla gene using ATPS-derived Taq DNA polymerase and commercial Taq DNA polymerase for 25 cycles, using the same template concentration in duplicates. ΔRn values were plotted against number of PCR cycles. Cyan-blue line corresponds to in-house ATPS-derived Taq DNA polymerase; dark blue line corresponds to commer-cial Taq DNA polymerase.
An aqueous two-phase system offers a promising alternative for industrially compatible purification procedures for recombinant proteins without compromising product quality or specificity.19,20 Partitioning in the aqueous two-phase system depends strongly on amino acid and peptide hydrophobicity, net charge, and the addition of salt to the system.21 It has been shown that negatively charged proteins largely partition in the salt layer, in contrast to positively charged proteins, which partition mostly into the polymer phase. From the data presented in this article, it is evident that, due to heat treatment, the majority of E. coli proteins do not partition into the salt layer along with Taq polymerase, in spite of the fact that they are acidic in nature and that Taq pol selectively localizes into the salt phase (as its pI is 6.0).
The observation of a significant increase in the specific activity of the Taq DNA polymerase (~eightfold) and ~117% recovery by means of a single-step purification protocol with ATPS (Table 1) appears interesting and is the first known report of its kind. Louwrier22 employed ethanol and potassium phosphate in an ATPS for removal of DNA from bacterial cells containing Taq pol to avoid non-specific PCR products. In the present study, we have eliminated most of the contaminating proteins in the interphase (>85%), together with host DNAs partitioned in the interphase and in the PEG phase of ATPS. This resulted in >95% pure Taq DNA polymerase protein in the salt phase in a single step with no detected nuclease activity (data not shown). Purity could be increased further yet by introducing a final chromatography step of choice.
Advantages of aqueous two-phase extraction include volume reduction, high capacity, and rapid separations. In order to avoid inconvenience during scale-up, dialysis step can effectively be replaced by techniques such as tangential flow filtration, or TFF (with a membrane of suitable molecular-weight cutoff) or use of a QuixStand™ hollow fiber system (GE Healthcare, Piscataway, New Jersey, USA). The currently described technique thus could be used as a sole step for purification of proteins at commercial manufacturing scale, as a cost-effective technique. Our observations are supported by the reports of easy scalability of ATPS23 for other proteins.
In conclusion, we report a method for purification of Taq DNA polymerase that is rapid and cost-efficient and could be applied to other polymerases, such as the Klenow fragment (DNA Pol I), and modifying enzymes such as T4 DNA ligase, T4 DNA polymerase, T4 polynucleotide kinase, etc. This method also appears to be a poten-
tial recovery technique for the purification of biomolecules, such as monoclonal antibodies, growth factors, and hormones, offering high selectivity and biocompatibility, with the simplicity of scale-up and continuous operation mode.24
ACKNOWLEDGMENTSThe authors thank Dr. Nagnath Mandi for making the Taq DNA
polymerase construct used in this study. Thanks are also due to Dr. Kamal Sharma, managing director at Lupin Limited, India, for encouragement and support.
REFERENCES
1. Chein A, Edgar DB, and Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127, 1550-1557 (1976).
2. Lawyer FC, Stoffel S, Saiki RK, and Chang SK. Isolation, characterization, and expression in E. coli of the DNA polymerase gene from Thermus aquaticus. J Biol Chem 264, 6427-6437 (1989).
3. Pluthero FG. Rapid purification of high activity Taq DNA polymerase. Nucleic Acids Res 23, 4850-4851 (1993).
4. Melissis S, Labrou NE, and Clonis YD. One-step purification of Taq DNA poly-merase using nucleotide-mimetic affinity chromatograpy. Biotechnol J 2, 121-132 (2007).
5. Engelke DR, Krikos A, Bruck ME, and Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem 191, 396-400 (1990).
6. Andersson E, and Hahn-Hagerdal B. Bioconversions in aqueous two-phase sys-tems. Enzyme Microbiol Technol 12, 242-254 (1990).
7. Tjerneld F, Berner S, Cajarville AE, and Johansson G. New aqueous two-phase system based on hydroxypropyl starch useful in enzyme purification. Enzyme Microbiol Technol 8, 417-423 (1986).
8. Walter H, Johansson G, and Brooks DE. Partitioning in aqueous two-phase sys-tems: Recent results. Anal Biochem 197, 1-18 (1991).
9. Esmanhoto E and Kilikian BV. ATPS applied to extraction of small molecules polycetides and simultaneous clarification of culture media with filamentous microorganisms. J Chromatogr B 807, 139-143 (2004).
10. Alstine KKV and Veide A. Aqueous two-phase systems—Methods and protocols. In: Methods in Biotechnology. Hatti-Kaul R (ed) 11, 251-258. Humana Press, Totawa, New Jersey, USA (1999).
11. Pathak SP, Sudha S, Sawant SB, and Joshi JB. New salt-polyethylene-glycol sys-tems for two-phase aqueous extraction. Chem Eng J 46:B31-B34 (1991).
12. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85 (1985).
Table 1 Purification of recombinant Taq DNA polymerase by ATPS and ammonium sulfate
Heat-treated cleared cell lysate containing Taq pol was divided into two equal fractions and subjected to ATPS and ammonium sulfate precipitation.
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300 INDUSTRIAL BIOTECHNOLOGY OCTOBER 2010
13. Grimm E and Arbuthnot P. Rapid purification of recombinant Taq DNA poly-merase by freezing and high temperature thawing of bacterial expression cul-tures. Nucleic Acids Res 23, 4518-4519 (1995).
14. Deshpnade A, Mandal G, Soorapaneni S, Prasad B, Kumar J, and Padmanabhan S. High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris. Biotechnol Lett 31, 811-817 (2009).
15. Lee C, Jaai Kim, Seung Gu Shin and Seokhwan Hwang. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123, 273-280 (2006).
16. Kepka C, Rhodin J, Lemmens R, Tjerneld F, and Gustavsson PE. Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system. J Chromatogr A 1024, 95 -104 (2004).
17. Kwon S, Jung Y, and Lim D. Proteomic analysis of heat-stable proteins in Escherichia coli. BMB Reports 41, 108-111 (2008).
18. Grunenwald H. Optimization of polymerase chain reaction PCR protocols. In: Methods in Molecular Biology, 2nd ed. Bartlett JMS and Stirling D (eds) 226 (89-100) Humana Press, Totawa, New Jersey, USA (2003).
19. Albertson PA. Partition of proteins in liquid polymer–polymer two-phase sys-tems. Nature 182, 709-711 (1958).
20. Guan Y, Lilley TH, Treffry TE, Zhou CL, and Wilkinson PB. Use of aqueous two-phase systems in the purification of human interferon-µ1 from recombinant Escherichia coli. Enzyme Microbiol Technol 19, 446-455 (1996).
21. Johansson HO, Persson J, and Tjerneld F. Thermoseparating water/polymer sys-tem: A novel one-polymer aqueous two-phase system for protein purification. Biotechnol Bioeng 66, 247-257 (1999).
22. Louwrier A. Nucleic acid removal from Taq polymerase preparations using an aqueous/organic biphasic system. Biotechniques 27, 444-445 (1999).
23. Cordes A and Kula MR. Large-scale purification of formate dehydrogenase. Methods Enzymol 228, 600-608 (1994).
24. Azevedo AM, Rosa PAJ, Ferreira IF, and Aires-Barros MR. Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol 27, 240-247 (2009).
