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Applications of BBa_K5387000

ALOX12B was cloned into pET-28a(+), which was confirmed by a whole plasmid sequencing.

The enzyme 12R-LOX was succesfully expressed and multiple measurements was performed.

Size Exclusion Chromatography

There were some shorter fragments after the purification with a Ni-NTA agarose column, hence a size exclusion chromatography was conducted to separate the fragments. The seperation was partly successful, due to the three largest fragments differed too little in size to separate completely with the columns available.

Mass Spectrometry

Using MALDI-TOF (Matrix Assisted Laser Desorption lonization -Time Of Flight), we could determine the molecular weight to 79,1 kDa. The actual weight is 82,5 kDa, but for larger proteins, such difference is excpected.

Dot Blot To ensure we had a His-tag from our pET-28a(+) plasmid, and thereby the correct enzyme expressed, we decided to do a dot blot to visualize the enzyme, as seen in figure 3.

Figure 3: Dot blot using Abcam HRP Anti-6X His tag® antibody, which binds and visualizes His-tags. A black dot confirms the presence of His-tags in a sample.

This further indicates that we in fact have synthesized and purified our enzyme!

Circular Dichroism

To investigate the secondary structure of the enzyme, a circular dichroism measurement was performed. The results from figure D, which shows alpha-helical structure with the dips at 208 nm and 220,nm, was further analyzed with the BestSel analysis program based on the studie [1] gives an estimated secondary structure of 64,7% a-helical and 35,3% beta-sheet structure of the enzyme. The estimated structure aligns with the generated AlphaFold structure [2] of 12R-LOX with an average model confidence of more than 90%.

Figure C: Spectrum from circular dichroism assessing secondary structure. The two dips, at 208 nm and 220 nm, indicates alpha helical structure in the protein, which coincides with the AlphaFold predicted structure.

Fluorescence Spectroscopy

To show whether 12R-LOX is folded, we did a wavelength scan for emission from our enzyme. The result is shown in figure X below, which clearly shows the emission peak at 340 nm. Tryptophan emits at shorter wavelengths when buried in a hydrophobic core of a protein [3] and at longer wavelengths (340-355 nm) when in a hydrophilic environment. The different Trp have different chemical surroundings, but emission the spectra clearly shows emission at 330-340 nm, hence indicating a folded protein.

Figure X: Wavelength scan of Tryptophan, showing a clear peak at 340 nm.

Nano Differential Scanning Fluorimetry

Foldedness

Discovered the two Tm of the enzyme at approximately 45°C and 57°C, as seen in "figure D" below. After adding dithiothreitol to reduce the disulfide bonds, we also discovered that the enzyme seem to have stabilizing disulfide bonds due to the loss of peaks and thereby a structure to unfold when dithiothreitol is present.

Figure D: nanoDSF spectra showing the ratio between 350 nm and 330 nm. The blue lines shows a representative graph from all duplicates with only 12R-LOX, and the orange line is 12R-LOX with dithiothreitol present.

Co-Factors

Encapsulation in liposomes

We encapsulated 12R-LOX in liposomes using thin film hydration and extrusion, the same way we encapsulated aeBlue (LÄNK). The result from aeBlue suggested encapsulation, and since the surrounding evidence and method used is identical, we can thereby assume 12R-LOX is encapsulated as well.

DLS bild

Fluorescence bild på liposomer, på 12R-LOX färglös och aeBlue som knotter.

References

[1] https://www.pnas.org/doi/10.1073/pnas.1500851112

[2] https://www.uniprot.org/uniprotkb/O75342/entry

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