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 seperate the fragments. The seperation was partly succesfull, due to the three largest fragments differed too little in size to seperate completely with the columns available.

Circular Dichroism

To investigate whether the enzyme was correctly folded, a circular dichroism measurement was performed. The results from "figure C", 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%.

NanoDSF

Discovered the two Tm of the enzyme at approximately 45°C and 57°C, as seen in "figure D" below. <left>

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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