Paul.xu at 19:19, 1 August 2014

2014-08-01T19:19:27Z

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Single-Step Purification of Recombinant Thermus aquaticus DNA Polymerase Using DNA-Aptamer Immobilized Novel Affinity Magnetic Beads 

DNA aptamer [Thiol-T-Pol-Apta] specific for Taq- polymerase was obtained from Thermo Electron, (Dreieich, Germany). The sequence of DNA-aptamer for Taq-polymerase enzyme was 5′-HS-(CH 2 ) 6 -TTTTTTGGCGGA-GCGATCATCT- CA-GAGCATTCTTAG-CGTTTTGTTCTT-GTCTATGA. Am- ersham E. coli GST-Gene Fusion Expression system was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Isopropyl β- D -thiogalactopyranoside (IPTG), polyvinyl alcohol (PVA), toluene, acetonitrile (ACN), and trifluoroacetic acid (TFA) were supplied from Sigma (St Louis, MO) and used as received. Methyl methacrylate (MMA), glycidyl methacrylate (methacrylic acid 2,3-epoxypropyl isopropyl ether; GMA), EGDMA, and R-R′-azoisobisbutyronitrile (AIBN) were obtained from Fluka (Switzerland), and the monomers were distilled under reduced pressure in the presence of hydroquinone and stored at 4 °C until use. All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). The water used in the present work was purified using a Barnstead system (Dubuque, IA). 

 

Preparation of Affinity Magnetic Beads. The magnetic beads were prepared in two sequential steps. In the first step, ferric-poly(GMA-MMA-EGDMA) beads were prepared via suspension polymerization, which was carried out in an aqueous dispersion medium containing FeCl 3 (0.3 M, 400 mL, used as a precursor for the thermal iron oxide precipitation in the beads). The organic phase contained GMA (7.5 mL), MMA (7.5 mL), EGDMA (7.5 mL, as cross-linker), and 5.0% PVA (20 mL, as stabilizer) were mixed together with 0.2 g of AIBN as initiator in 20 mL of toluene. The polymerization reactor was placed in a water bath and heated to 65 °C. The reactor was then equipped with a mechanical stirrer, nitrogen inlet, and reflux condenser. The polymerization reaction was maintained at 70 °C for 2.0 h and then at 80 °C for 1.0 h. After the reaction, the resultant beads were filtered under suction and washed with distilled water and ethanol. In the second step, magnetic poly(GMA-MMA-EGDMA) beads were prepared by conventional coprecipitation reaction of iron oxide in the beads. For the coprecipitation reaction, 5.0 g og FeCl 2 was dissolved in purified water (100 mL) and then

transferred into a reactor containing ferric-poly(GMA-MMA- EGDMA) beads (15 g) in NH 3 /H 2 O (50 mL, 25% w/v). The reactor was equipped with reflux condenser and refluxed under nitrogen atmosphere at 40 and 50 °C for 2 h and then at 90 °C for 2 h with continuous stirring. Finally, the synthesized magnetic beads were separated from the reaction medium, washed in ethanol solution (70%; 250 mL) for 3 h, and then washed with purified water. During the thermal precipitation reaction, the epoxy groups of the magnetic beads were also converted into amino groups via ammonolysis reaction due to the presence of ammonia in the medium (27). Glutaric dialde- hyde activation of the amino groups of the supports was carried out as described previously (28). A schematic representation of the chemistry of the preparation of magnetic affinity beads is presented in Figure 1. DNA-thiol-aptamer specific for Taq-polymerase was im- mobilized covalently via its thiol group to the activated magnetic poly(GMA-MMA-EGDMA) beads. The beads were washed six times with phosphate buffer (1.0 mL, 50 mM; pH 8.0) by a magnetic separation technique (i.e., the beads were trapped in a magnetic field) and after the aspiration of the solution, they were then resuspended in fresh buffer solution. The coupling reaction was initiated by addition of DNA-aptamer to the activated magnetic beads suspension (50 mg of magnetic beads in a total of 500 μL reaction medium), which was then vortexed thoroughly and incubated for different time points and conditions (pH between 4.0 and 11.0; DNA-aptamer concentration between 50 and 500 μg/mL) under 50 rpm shaking. After 6 h of reaction, the immobilization medium was then removed and DNA- aptamer immobilized magnetic beads were washed three times with phosphate buffer (1.0 mL, 50 mM, pH 8.0). The amounts of DNA-aptamer in the initial and final mixture were measured at 260 nm by using a double beam UV-vis spectrophotometer (model 1601, Shimadzu, Tokyo, Japan). 

 

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Preparation of Ferrites /Fe 3 O 4 The FeCl 3 .6H 2 O (5.4 g) and urea (3.6 g) were dissolved in water (200 mL) at 85 to 90 o C for 2 h. The solution turned brown in color. The resultant reaction mixture was cooled to room temperature to which was added FeSO 4 .7H 2 O (2.8 g) and then 0.1 M NaOH until pH 10. The molar ratio of Fe (III) to Fe (II) in the above system was nearly 2.00. The obtained hydroxides were treated by ultrasound in the sealed flask at 30 to 35 o C for 30 min. After ageing for 5 h the obtained black powder of Fe 3 O 4 was washed, and dried under vacuum.

Iron oxide particles were synthesized as described by Dallas et al., (2007) with slight modification. Briefly, 1.25 g of FeSO 4 .7H 2 O and 0.6 g KOH (Merck, India) was dissolved in 30 ml H 2 O of prefixed temperature (50°C). The mixture was refluxed for 1 h. The as- prepared magnetic solids were insoluble in aqueous or organic media. They were isolated by centrifugation, washing several times with water and drying. The particles thus obtained exhibited a strong magnetic response. 

Ferric chloride, 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), sodium hydroxide, acetic anhydride, iron(II) chloride tetrahydrate 97%, iron(III) chloride hexahydrate 99%, nitric acid, (3-aminopropyl)trimethoxysilane, 1-butanol, octane, toluene, metha- nol, L -aspartic acid, tetramethylammonium bromide, 11-mercap- toundecanoic acid, and sodium tetrahydridoborate were obtained from Sigma-Aldrich (St. Louis, MO), 

 

Preparation of Fe 3 O 4 Magnetic Nanoparticles. Magnetic nanoparticles Fe 3 O 4 were prepared by the hydrothermal copre- cipitation of ferric and ferrous ions using NaOH as the base, described by Kouassi et al. 27 Typically iron(II) chloride and iron- (III) chloride (1-2) were dissolved in Nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25 °C) by adding 1 M NaOH at a constant of pH 10. The precipitates were heated at 80 °C for 35 min under continuous mixing and washed four times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of 

trength greater than 20 megaoersted (MOe), and the particles were finally dried in a vacuum oven at 70 °C. Surface Modification of Fe 3 O 4 Magnetic Nanoparticles with APTMS. Magnetic nanoparticles (1 g) were washed with 99.5% methanol and twice with Nanopure water and soaked in 10 mL of 3 mM APTMS solution in a toluene/methanol (1:1 v/v) mix. The suspension was then transferred into a three-necked flask with a water-cooled condenser and temperature controller with a nitrogen gas flow at 80 °C for 20 h under vigorous stirring. Silanization was bound to occur at the surfaces of the particles bearing hydroxyl groups, which in the presence of an organic solvent results in the formation of an APTMS coating with a large density of amines. 20 The particles were recovered by applying an external magnetic field after the silanization process and washed three times with methanol and dried at 50 °C in a vacuum oven. LAA Functionalization of Magnetic Nanoparticles. One gram of magnetic nanoparticles was immersed in 50 mL of 0.1 M LAA solution prepared in nitric acid at pH ∼2. The mixture was sonicated for 15 min and vigorously stirred for 10 h at room temperature. An external magnetic field was applied to recover the particles and washed two times with Nanopure water. The process is expected to ensure the chemisorption of LAA, bearing carboxyl and amino groups on the surface of the particles. 

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Magnetic beads - Revision history

Paul.xu at 19:19, 1 August 2014