Biomod/2013/Xiamen/XMU-Nanobiocat/Res Med

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1.Construction of BL21(DE3)pET28a-bis-His tag GDH-NOX
BL21(DE3)pET32a-GDH and BL21(DE3)pET32a-NOX was from Institute of Biochemical Engineering of Xiamen University. First, the gene of GDH and NOX was amplified by PCR. The primers were listed followed:
(Forward primer of GDH was added with "CACCACCACCACCACCAC" which encoded six Histidines)
The plasmid pET32a-GDH and pET32a-NOX was extracted using Plasmid Mini Kit 1 purchased from OMEGA bio-tek.

The electrophoresis results accorded with the theoretical size of each gene. Then the PCR products were recovered and used for SOE-PCR.

A PCR product of about 2500 bp was amplified and the size was accorded with the theoretical size of GDH-NOX gene

pET28a-bis-His tag GDH-NOX plasmid was extracted and digested with NcoI and XhoI to roughly check if the plasmid was correctly constructed.

It was showed that one digested product was about 2500bp and the other product was above 5000 bp. The result fitted the theoretical length of pET28a-bis-His tag GDH-NOX and pET28a fragment.
pET28a-bis-His tag GDH-NOX plasmid was then sequenced by SANGON BIOTECH using T7 promoter and T7 terminator as primers. Alignments were performed using the program CLUSTAL W.
The plasmid was then transformed into E. coli BL21(DE3) for expression of bis-His tag GDH-NOX.

2. Expression and purification of bis-His tag GDH-NOX
Expression of bis-His tag GDH-NOX was induced by IPTG in concentration of 1mM for 4 hours. After induction, E.coli biomass was harvested by centrifugation (8,000 g, 10 min, 4℃) and disrupted by ultrasonication (200W, 5s on, 5s off, 24 times). Debris of bacterial cells were removed by ultracentrifugation at 14,000 g for 15 min at 4℃.
Purification was conducted by AKTA Prime Plus(GE Healthcare).
The crude cell extract was applied on the Histrap HP column (GE Healthcare,5ml). After sample was loaded, the column was washed 20 times of column volume wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) and then eluted 5 times of column volume with elution buffer ( 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4). The fractions containing pure protein were collected and desalted with PD-10 column (GE Healthcare). The resulting enzyme solution was used for further experiment.

The size of purified protein was about 110 kDa shown by SDS-PAGE. The protein concentration of bis-His tag GDH-NOX was 90.5 mg/ml. GDH and NOX enzyme activity was 50.5 U/mg and 88.7 U/mg respectively.

3. Assembly of enzyme nanoring mediated by nickel ion
3.1 Nickel ion mediated Assembly
bis-His tag GDH-NOX obtained from PD-10 desalting column was added with 25 mM, 50 mM, 100 mM nickel sulfate and incubated at 37℃ for 30 min. We found that nickel in these three concentrations can instantly make enzyme solution turning turbid.

With the addition of nickel sulfate, one bis-His tag GDH-NOX could combine more than two enzyme molecules due to its bis-His tags, while GDH only formed dimers. Therefore, the size of solutes in left vial is larger than that in right vial, the left vial was turbid while the right was transparent.
3.2 Dynamic Light Scattering (DLS) analysis
To evaluate the effect of nickel sulfate mediated enzyme assembly, DLS was used to measure the size of the assemble structure of bis-His tag GDH-NOX.
Samples with three different nickel sulfate concentrations and a blank sample without nickel sulfate were prepared and analyzed by Zetasizer-Nano ZS90 (Malvern Instruments) for DLS analysis. The figures of size distribution by number were as following.

* When 100 mM NiSO4 was added, Peak 2 (4451 nm) appeared.
The result showed that the average size of bis-His tag GDH-NOX was 84.68 nm. Addition of nickel sulfate could assemble bis-His tag GDH-NOX into multi-enzyme complex which had diameter nearly 1μm. As the concentration of nickle sulfate increased, the size of the multi-enzyme complex increased.
3.3 Scanning Electron Microscope (SEM) analysis
The morphologies of the nanoparticle formed by nickel ion mediated bis-His tag GDH-NOX assembling were observed at different nickel concentrations increasing from 25 mM to 100 mM were observed. The result accorded with the size date from DLS experiment.

Figure 3.3 and Figure 3.4 suggested that the following mechanism for the formation of enzyme nanoparticle rather than nanoring. Since His-tag has six Histidines then it could combine with more than one nickel ion. In the first step, the ability to combine more nickel ions made it possible for enzyme to assemble together to formed triple or quadruple structure and further formed a enzyme nanoparticle. In the second step, the enzyme nanoparticle could pile up like grape probably through the next step interaction between the His-tag on the surface of the enzyme nanoparticles and free nickel ions.

4. Tanning experiment
4.1 DHA synthesis by purified enzyme
The ability of bis-His tag GDH-NOX to produce DHA was tested for 70 hours. The DHA concentration was monitored and the experimental result was as follows.

After reaction for 57 hours, the highest concentration of DHA was 0.26 mg/ml which was not sufficient for tattoo purposes.
4.2 DHA synthesis by crude enzyme
Because crude enzyme often had better stability, crude bis-His tag GDH-NOX was used for DHA synthesis. The DHA yield was higher than that used purified enzyme.

4.3 Sunless Tanning
In order to test the ability of bis-his tag GDH-NOX, a series of Sunless tanning experiments were performed using purified and crude bis-His tag GDH-NOX and 5% DHA. Several hours after applying enzyme or DHA on tryptone containing filter paper, the tanning pattern could be observed on the filter paper which was applied with 5% DHA. Unfortunately the purified and crude enzyme could not caused an visible change on the filter paper. Some sample patterns (blank runs using 5% DHA during tanning experiments) are shown on the Figure 4.3 , Figure 4.4 and Figure 4.5.

1 Genetic Manipulation
1.1 Plasmid isolation
      Plasmid Mini Kit 1 (OMEGA bio-tek) was used to isolate plasmid DNA.

1.2 Enzyme Digestion

      Reaction condition: 37℃,1h.
      NcoⅠand XhoⅠwas purchased from Takara.
1.3 Polymerase Chain Reaction (PCR)
      All the PCR was performed using Takara Ex Taq DNA polymerase.
      The PCR conditions for amplifying different DNA were listed in "Result" part.

1.4 Agarose Gel Electrophoresis of DNA
      Gel Electrophoresis was running in 1.5% agarose gel, at 140 V for 20-30 min. Takara 500 bp DNA ladder and 1K bp DNA ladder was used to evaluate the product size.
1.5 DNA fragment Recovery
      E.Z.N.A.Gel Extraction Kit (OMEGA bio-tek) was used to recover DNA fragment from agarose gel.
1.6 DNA Ligation (Kit: TAKARA DNA Ligation Kit Ver.2.1 )
      Mix the vector DNA and the DNA fragment to prepare a 5-10μl DNA solution.
      Add an equalvolume solution 1 and mix.
      React for thirty minutes in 16℃.
1.7 Competent cell preparation and DNA Transformation
      Competent cell preparation and DNA transformation was performed following the manual of Competent cell preparation kit (Takara).

2. Gene expression and enzyme purification
2.1 Express bis-His tag GDH-NOX gene
      The bis-His tag GDH-NOX gene was expressed in BL21(DE3) E.coli. The culture was grown at 37℃ for 2h to reach a OD600=0.6-0.8. The enzyme expression was induced by adding 1 mM isopropylthiogalactoside (IPTG) at 30℃ for 4 h.
2.2 Extraction of intracellular enzyme
      Cells were harvested by centrifugation. 1 g of cell pellet was suspended with 5 ml of lysis buffer(20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 2mM Dithiothreitol (DTT), pH 7.4) , and the cells were broken by untrasonication. The broken cells were centrifuged and the debris was discarded.

2.3 Enzyme purification
      The cell-free extract was loaded on Histrap HP (5ml, GE Healthcare). The column was washed with 20 column volume of binding buffer(20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and then eluted with 5 column volume of elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4). The enzyme activity of fractions were then measured and the fraction containing targeting enzyme was desalted with PD-10 column (GE Healthcare).

      Preparing SDS-PAGE gels (1 mm).
      Add denaturing loading buffer in enzyme samples, and apply 20 μl samples into gel.
      Run the gel at 80 V for spacer gel, 140 V for resolving gel. Stop electrophoresis until the bromophenol blue band reached the bottom of the resolving gel.
      Remove the glass plates and stain the gel with Coomassie Brilliant Blue R-250.
3. Nickel ion mediated enzyme assembly
      Dissolve the fresh enzyme in 20 mM PBS buffer containing 50 mM NaCl.
      Add NiSO4 with different concentrations and maintain the assemble tube in 37℃ for 30 min.

4. In situ synthesis of DHA and sunless tanning
4.1 In situ synthesis of DHA
      Typical synthesis reaction contained 200 ul purified bis-His tag GDH-NOX and 800 ul tanning mix (1M glycerol, 1mM NAD+ dissolved in PBS buffer, pH 7.4). Reaction condition was 37℃, 200 rpm.
4.2 Sunless tanning
      To simulate the DHA tanning on human skin, we need to find a suitable test material which contains amines or amino groups to be reacted with DHA. Tryptone was found to be an inexpensive and convenient material for testing DHA tanning.
      First, tryptone was dissolved in H2O with the concentration of 50 g/L. The solution and filter paper was then placed in Petri dish and maintained in 80℃ oven until it formed a layer of solid tryptone. After the tryptone filter paper was ready, DHA solution or enzyme solution contain glycerol and NAD+ was applied on the filter paper and the tanning results were recorded.

5. Characterization and analytic approaches
5.1 Enzyme activity (GDH/NOX)
      NADH oxidase (NOX):

      Glycol dehydrogenase (GDH):

      These two reactions were conducted at 37℃ in a 96-well plates in Multiskan FC (Thermo Scientific). Once the reaction started, OD340 was measured every 10 second for 6 min.
      The activity was defined as the number of micromoles of NADH consumed (or produced) in 1 min by 1 mg of enzyme( (μmol/min/mg).

5.2 Determination of protein content (Bradford)
      Prepare a series of BSA protein standards of 0, 10, 20, 40, 60, 80 and 100 µg/mL. Also prepare dilutions of the unknown sample to be measured.
      Add 20 µL to each of the above to a 96-well plate.
      Add 200 µL Coomassie Blue solution.
      Wait for 5 minutes and measure OD570 in Multiskan FC (Thermo Scientific).
      Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

5.3 Determination of DHA concentration
      Preparation of phosphomolybdic acid.
      7 g Molybdic acid was dissolved in 40 ml NaOH solution (100 g/L), then added 40 ml H2O to the solution and heated in boiling bath for 20 min. After the solution was cooled, 25 ml concentrated phosphoric acid was added.
      Draw standard curve of DHA.
      1 ml 0.1, 0.15, 0.2, 0.25 and 0.4 mg/ml DHA was added to 1 ml above phosphomolybdic acid, respectively. Then heated them in boiling bath for 15 min and the color of the solution was turned blue. Added 100 ml solution to 3 ml H2O and measured the OD value to draw the standard curve of DHA.
      Measure the concentration of DHA.
      Added 100 µl sample to 100 µl above phosphomolybdic acid and heated them in boiling bath for 15 min. Then took 100 µl heated solution to 3 ml H2O and measured OD630.

5.4 Dynamic Light Scattering (DLS)
      DLS was performed using Zetasizer-Nano ZS90 (Malvern Instruments) in public instrument platform of Department of Chemical Biology  of Xiamen University.
5.5 Scanning Electron Microscope (SEM)
SEM was performed using Hitachi S-4800 High resolution SEM in public instrument platform of College of Chemistry and Chemical Engineering of Xiamen University.

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