Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction
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Notes & Tips Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction Rui Ma a,1 , Jun Zhao b,1 , Hui-Cong Du a , Shuang Tian a,c , Li-Wen Li a,⇑ a Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an 710069, China b Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an 710032, China c Department of Obstetrics and Gynecology, General Hospital of Chinese PLA (People’s Liberation Army), Beijing 100853, China article info Article history: Received 14 January 2012 Accepted 10 February 2012 Available online 25 February 2012 Keywords: Endotoxin removing Plasmid Triton X-114 abstract Triton X-114 temperature transition extraction has been considered to be a simple and cost-effective strategy to eliminate endotoxin from plasmid preparations. However, a repeated cooling–heating process may promote the degradation of plasmid DNA. Based on the finding that the cloud point of Triton X-114 solution increases substantially in the presence of small amounts of sodium dodecyl sulfate (SDS) and that electrolytes decrease the cloud point of Triton X-114–SDS solution drastically, we designed a Triton X-114 isothermal extraction method for removing endotoxin from plasmid samples and found that it has the same endotoxin removal efficiency when compared with the temperature transition extraction method. Ó 2012 Elsevier Inc. All rights reserved. Plasmid has been widely accepted as a safe gene delivery vector in the field of gene therapy and gene immunization. Although many strategies for large-scale preparation of plasmid have been well established, removal of endotoxin contaminants remains an expensive and/or labor-intensive procedure. Several methods to eliminate endotoxin from plasmid preparations have been re- ported, including cesium chloride density gradient centrifugation, anion exchange chromatography, polymyxin B chromatography, and Triton X-114 extraction [1,2]. In view of both convenience and efficiency, Triton X-114 extraction was evaluated as a rapid and effective method for endotoxin removal from plasmid samples. However, a repeated cooling–heating process may promote plas- mid DNA degradation. In this article, we report a novel Triton X-114 isothermal endotoxin extraction method established based on the fact that sodium dodecyl sulfate (SDS) 2 and NaCl have a converse effect on the cloud point of Triton X-114 solution. Silicon dioxide, NaI, Triton X-114, SDS, and NaCl were pur- chased from Sigma. Homogeneous Triton X-114 was prepared by three 0/30 °C temperature cycles. TXS solution (6% [w/v] Triton X-114 and 3.0% [w/v] SDS) was prepared by dissolving 12.0 g of Triton X-114 and 6.0 g of SDS in 180 ml of distilled water with magnetic stirring and bringing to a final volume of 200 ml. Oil red could be added to TXS solution to act as an indicator. To prepare silicon dioxide suspension, 10 g of silicon dioxide was suspended thoroughly in 100 ml of sterile distilled water and centrifuged at 1000g at room temperature for 15 min. The supernatant from the top was discarded, and the silicon dioxide was washed two more times. After washing, the silicon dioxide was resuspended in 20 ml of distilled water for further use. Plasmid was prepared using the method reported previously with minor modifications [3–5]. Escherichia coli DH5a cells harbor- ing plasmid pEGFP-N3 were grown in Luria–Bertani (LB) medium in the presence of 30 lg/ml kanamycin. Cell paste from 500-ml overnight cultures was obtained by centrifugation and resuspended in 10 ml of buffer 1 (50 mM glucose, 25 mM Tris– HCl, 10 mM ethylenediaminetetraacetic acid [EDTA], and 100 mg/ L RNase A, pH 8.0), lysed for 5 min by the addition of 20 ml of buf- fer 2 (0.2 M NaOH and 1% SDS), and neutralized by 15 ml of buffer 3 (3 M potassium acetate, pH 4.8). After centrifugation, solid NaCl was added to the supernatant to obtain a final concentration of 2.5 M. Then 2 ml of silicon dioxide suspension was added and mixed well with continuous agitation for 5 min. After centrifuga- tion, silica was washed twice with buffer 4 (60% ethanol, 10 mM Tris–HCl, 100 mM NaCl, and 1 mM EDTA). Finally, distilled water was added to dissolve the plasmid DNA from silicon dioxide, and the concentration of plasmid DNA sample was adjusted to 1.0 mg/ml. Endotoxin was removed by the method of Triton X-114 temperature transition extraction or isothermal extraction. Plasmid DNA in the final aqueous phase was precipitated with an equal volume of isopropanol at room temperature. After washing 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.02.015 ⇑ Corresponding author. E-mail address: [email protected] (L.-W. Li). 1 These authors contributed equally to this work. 2 Abbreviations used: SDS, sodium dodecyl sulfate; LB, Luria–Bertani; EDTA, ethylenediaminetetraacetic acid; LPS, lipopolysaccharide. Analytical Biochemistry 424 (2012) 124–126 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Transcript of Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction
Analytical Biochemistry 424 (2012) 124–126
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier .com/locate /yabio
Notes & Tips
Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction
Rui Ma a,1, Jun Zhao b,1, Hui-Cong Du a, Shuang Tian a,c, Li-Wen Li a,⇑a Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an 710069, Chinab Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an 710032, Chinac Department of Obstetrics and Gynecology, General Hospital of Chinese PLA (People’s Liberation Army), Beijing 100853, China
a r t i c l e i n f o a b s t r a c t
Article history:Received 14 January 2012Accepted 10 February 2012Available online 25 February 2012
Keywords:Endotoxin removingPlasmidTriton X-114
0003-2697/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ab.2012.02.015
⇑ Corresponding author.E-mail address: [email protected] (L.-W. Li).
1 These authors contributed equally to this work.2 Abbreviations used: SDS, sodium dodecyl sulfat
ethylenediaminetetraacetic acid; LPS, lipopolysaccharid
Triton X-114 temperature transition extraction has been considered to be a simple and cost-effectivestrategy to eliminate endotoxin from plasmid preparations. However, a repeated cooling–heating processmay promote the degradation of plasmid DNA. Based on the finding that the cloud point of Triton X-114solution increases substantially in the presence of small amounts of sodium dodecyl sulfate (SDS) andthat electrolytes decrease the cloud point of Triton X-114–SDS solution drastically, we designed a TritonX-114 isothermal extraction method for removing endotoxin from plasmid samples and found that it hasthe same endotoxin removal efficiency when compared with the temperature transition extractionmethod.
� 2012 Elsevier Inc. All rights reserved.
Plasmid has been widely accepted as a safe gene delivery vectorin the field of gene therapy and gene immunization. Althoughmany strategies for large-scale preparation of plasmid have beenwell established, removal of endotoxin contaminants remains anexpensive and/or labor-intensive procedure. Several methods toeliminate endotoxin from plasmid preparations have been re-ported, including cesium chloride density gradient centrifugation,anion exchange chromatography, polymyxin B chromatography,and Triton X-114 extraction [1,2]. In view of both convenienceand efficiency, Triton X-114 extraction was evaluated as a rapidand effective method for endotoxin removal from plasmid samples.However, a repeated cooling–heating process may promote plas-mid DNA degradation.
In this article, we report a novel Triton X-114 isothermalendotoxin extraction method established based on the fact thatsodium dodecyl sulfate (SDS)2 and NaCl have a converse effect onthe cloud point of Triton X-114 solution.
Silicon dioxide, NaI, Triton X-114, SDS, and NaCl were pur-chased from Sigma. Homogeneous Triton X-114 was prepared bythree 0/30 �C temperature cycles. TXS solution (6% [w/v] TritonX-114 and 3.0% [w/v] SDS) was prepared by dissolving 12.0 g ofTriton X-114 and 6.0 g of SDS in 180 ml of distilled water with
ll rights reserved.
e; LB, Luria–Bertani; EDTA,e.
magnetic stirring and bringing to a final volume of 200 ml. Oilred could be added to TXS solution to act as an indicator.
To prepare silicon dioxide suspension, 10 g of silicon dioxidewas suspended thoroughly in 100 ml of sterile distilled waterand centrifuged at 1000g at room temperature for 15 min. Thesupernatant from the top was discarded, and the silicondioxide was washed two more times. After washing, the silicondioxide was resuspended in 20 ml of distilled water for furtheruse.
Plasmid was prepared using the method reported previouslywith minor modifications [3–5]. Escherichia coli DH5a cells harbor-ing plasmid pEGFP-N3 were grown in Luria–Bertani (LB) mediumin the presence of 30 lg/ml kanamycin. Cell paste from500-ml overnight cultures was obtained by centrifugation andresuspended in 10 ml of buffer 1 (50 mM glucose, 25 mM Tris–HCl, 10 mM ethylenediaminetetraacetic acid [EDTA], and 100 mg/L RNase A, pH 8.0), lysed for 5 min by the addition of 20 ml of buf-fer 2 (0.2 M NaOH and 1% SDS), and neutralized by 15 ml of buffer3 (3 M potassium acetate, pH 4.8). After centrifugation, solid NaClwas added to the supernatant to obtain a final concentration of2.5 M. Then 2 ml of silicon dioxide suspension was added andmixed well with continuous agitation for 5 min. After centrifuga-tion, silica was washed twice with buffer 4 (60% ethanol, 10 mMTris–HCl, 100 mM NaCl, and 1 mM EDTA). Finally, distilled waterwas added to dissolve the plasmid DNA from silicon dioxide, andthe concentration of plasmid DNA sample was adjusted to1.0 mg/ml. Endotoxin was removed by the method of TritonX-114 temperature transition extraction or isothermal extraction.Plasmid DNA in the final aqueous phase was precipitated with anequal volume of isopropanol at room temperature. After washing
Fig.1. (A) The mixture of TXS solution with plasmid sample was a clear andhomogeneous solution at room temperature. (B) Adding 5 M NaCl made themixture cloudy. (C) After centrifugation at 12,000g for 10 min, the mixture wasseparated as upper aqueous phase and lower Triton phase.
Notes & Tips / Anal. Biochem. 424 (2012) 124–126 125
the precipitate twice with 70% ethanol at 4 �C and air-drying,the plasmid was redissolved in sterile distilled water andquantified by a NanoView spectrophotometer (GE HealthCare). ABioWhittaker QCL-1000 Chromogenic Limulus Amebocyte Lysatetest was used to quantify endotoxin.
The traditional method of extraction of lipopolysaccharide (LPS)from DNA samples was carried out as that previously publishedwith minor modifications [1]. Briefly, Triton X-114 was added tothe plasmid sample to a final concentration of 1.0% (w/w). Thesolution was incubated in an ice bath with intermittent shakinguntil the solution became clear and homogeneous. Then the solu-tion was transferred to a 45 �C water bath for 10 min, and a two-phase system was separated by centrifugation at 37 �C for10 min. After discarding the bottom Triton X-114 phase, anothertwo cycles of treatment were performed with the top aqueousphase.
Triton X-114 isothermal extraction was performed as follows.After 1 volume of DNA sample was mixed with 0.2 volume ofTXS solution, the clear mixture was incubated at room temperaturefor 10 min. After adding 5 M NaCl to a final concentration of 1.0 Mand mixing well by hand shaking, the mixture was centrifuged at12,000g in a microcentrifuge for 10 min at room temperature.The upper aqueous phase was transferred to a fresh Eppendorftube, and the extraction procedure was repeated two more times.
Triton X-114 temperature transition extraction has been con-sidered as a simple and effective method of endotoxin removal.
Fig.2. After three rounds of Triton X-114 extraction, the endotoxin content in the platransition (A) and isothermal extraction (B).
At temperatures below its cloud point of 23 �C, Triton X-114 is mis-cible with aqueous solutions; at temperatures above 23 �C, thedetergent partitions into a separate phase. This phase partitioninghas been reported to be capable of extracting the lipophilic endo-toxin molecule from DNA preparations as well as aqueous proteinsolutions. However, Triton X-114 is too sticky to be manipulatedaccurately. In addition, repeated cooling–heating may damagethe plasmid DNA.
It has been reported that the cloud point of Triton X-114 solu-tion increases substantially in the presence of small amounts ofSDS and that the cloud point of TXS solution decreases drasticallywhen electrolytes are added [6,7]. In the current work, we investi-gated whether this characteristic of Triton X-114 could help us todesign an isothermal endotoxin extraction method. Mixing TXSsolution with plasmid sample, we got a clear and homogeneoussolution at room temperature (Fig. 1A). Adding 5 M NaCl madethe mixture cloudy (Fig. 1B). After centrifugation, the mixturewas separated as upper aqueous phase and lower Triton phase(Fig. 1C). To determine whether this method could remove endo-toxin efficiently, we performed both temperature transitionextraction and isothermal extraction on three different plasmidsamples. As we expected, the results showed that isothermalextraction has the same efficiency in removing endotoxin whencompared with temperature transition extraction (Fig. 2). In addi-tion, after three cycles of extraction, the endotoxin content in theplasmid samples was below 0.1 EU/6 lg DNA, which has been con-sidered as endotoxin free. Moreover, by using isothermal extrac-tion, the plasmid DNA recovery rates were 87.6 ± 3.8%, a littlehigher than when using temperature transition extraction(79.7 ± 2.6%).
In conclusion, we have provided a simple method to removeendotoxin from laboratory-scale preparation of plasmid DNA byTriton X-114 isothermal extraction. In addition, isothermal extrac-tion has the same efficiency as temperature transition extraction ineliminating endotoxin. However, the Triton X-114 isothermalextraction method could not be applied to remove endotoxin fromprotein solution because SDS has a hydrophobic tail that interactsstrongly with protein, and this could make significant changes inprotein conformation.
Acknowledgments
This work was supported by the National Natural Science Foun-dation of China (grant 30873161 to L-W. Li and grant 81001448 toS. Tian) and by the Foundation of Shaanxi Educational Committeeof China (grant 2010JS099 to L-W. Li).
smid samples was below 0.1 EU/6 lg DNA (endotoxin free) by both temperature
126 Notes & Tips / Anal. Biochem. 424 (2012) 124–126
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