Part:BBa_K5387000:Experience

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Applications of BBa_K5387000

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.

Figure 1: SDS-PAGE after Ni-NTA agarose column but before size exclusion chromatography.

Figure 2: SDS-PAGE after size exclusion chromatography.

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.

NanoDSF

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.

Wavelength Scan

References

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

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

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