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Applications of BBa_K5387000

Cloning the Gene into a Plasmid

We began by ordering the human gene, ALOX12B, and primers with restriction sites to amplify the gene and add restriction sites to the end of it. The restriction sites were used to cleave the fragment with restriction enzymes to form blunt ends, which were ligated together with a cleaved pET-28a(+) to form our plasmid with the desired gene. The plasmid was transformed into NEB® 5-alpha competent E. coli for amplification, where a colony and plasmid screening was performed to see which of the ligations were successful. As shown in figure 1 below, we had two successful colonies:

Figure 1. An agarose gel, 1%, showing the screening for our inserted gene in the plasmid. The two band indicate ALOX12B was successfully cloned into a vector. As shown, the molecular weight is between 2 kb and 3 kb.

For further verification, the whole plasmid was sent for sequencing, which confirmed that the gene had successfully been cloned into pET-28a(+).

Expression and Purification

We later transformed the plasmid into BL21 competent E. coli for expression when induced with IPTG. We started a large-scale expression and purification of 12R-LOX, the enzyme encoded by the gene ALOX12B. The SDS-PAGE showed protein close to the correct Mw for the enzyme, though with multiple bands, as seen in figure 2. The correct molecular weight with the His-tag would be ~82.5 kDa, whereas the gel only shows ~70 kDa. There are also multiple fragments below the correct Mw, indicating that the enzyme might be degraded in E. coli, or not purified enough in the Ni-NTA agarose column.

Figure 2. SDS-PAGE of multiple steps during protein purification. From the left: Molecular weight ladder, cell pellet, sample eluded from the Ni-NTA column, flow through while adding the sample to the Ni-NTA column, a blank well, and to the right, TTR protein containing a His-tag used as a positive control.

Dot Blot

To ensure we had a His-tag from our pET-28a(+) plasmid, and thereby expressed the correct enzyme, 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 indicates that we have synthesized our enzyme!

Size Exclusion Chromatography

From here, we wanted to purify the enzyme from the smaller fragments seen in figure 2. We thereby did a size exclusion chromatography (SEC) to separate the bands. All runs to purify 12R-LOX yielded the chromatogram, or close to identical, as seen in figure 4. The chromatogram shows that the separation worked very well, as confirmed in figure 5. In all runs, fractions from the first peak were pooled and concentrated, but unfortunately, we did not manage to separate the three heaviest fragments.

Figure 4. Size exclusion chromatography of 12R-LOX with the column Cytiva Superdex™ 75 increase 10/300 GL. Measured UV at 280 nm to see when protein was eluted. The chromatogram shows the first time the SEC was run, but all following runs on this column yielded a similar elution pattern.

Figure 5. SDS-PAGE with a ladder in the first well and the three largest bands from one of the fractions after SEC in the second well, showing a successful separation from the smaller fragments.

Mass Spectrometry

Since the bands from SDS-PAGE of 12R-LOX appeared between 50-75 kD (according to the molecular weight marker) and we expected an 80 kD protein we moved on to mass spectrometry. To further verify the expression of our enzyme, we performed a Matrix Assisted Laser Desorption lonization -Time Of Flight (MALDI-TOF). As seen in figure 6, the main peak is located at 79.1 kDa, with charge +1 and +2 at half mass, at 39.7 kDa. There are also some other minor peaks in the spectrum, likely from truncated 12R-LOX or impurities. The deviation from the actual molecular weight, 82519 Da, is likely due to the size of the protein. The larger the protein, the more difficult it is to get it to fly in the MALDI-TOF [1] and get an accurate molecular weight.

Figure 6. Spectra from MALDI-TOF of 12R-LOX. The main peak with charge +1 is at 79 132 Da, and the peak with charge +2 is at 39 782 Da, corresponding to a molecular weight of 79564 Da.

Circular Dichroism Spectroscopy

Using the samples from the first peak in the size exclusion chromatography, we started to perform characterization and stability measurements on 12R-LOX. Circular Dichroism (CD) spectroscopy was performed to quantify the secondary structure of our enzyme and to give further verification of it being folded and 12R-LOX. The experiment gave clear results, with dips, as can be seen in figure 7, located at 208 and 220 nm, clearly indicating a mainly alpha helical structure of the enzyme. The resulting graph was further analyzed with the BeStSel™ analysis program [2] giving an estimated secondary structure of 64,7% α-helical and 35,3% β-sheet structure of the enzyme, as seen in figure 8. The estimated structure percentage coincides with the generated AlphaFold structure of 12R-LOX [4-5], seen in figure 9, with an average model confidence of more than 90%. The similarity of the results with the simulation further strengthens that we have successfully produced 12R-LOX.

Figure 7. Wavelength scan measuring molar ellipticity for 12R-LOX, showing two dips at 208 and 222 nm, indicating a primarily ɑlpha-helical structure of the enzyme.

Figure 8. Estimated secondary structure of 12R-LOX calculated with the BeStSel™ analysis program.

12R-LOX Enzyme Structure

12R-LOX contains 13 tryptophans (Trp) [3], as seen in figure 9, which is very beneficial when performing different measurements since it has fluorescent properties and is therefore very sensitive.

Figure 9. Alpha-Fold2 structure model [4-5] of 12R-LOX visualized in PyMol [6], with tryptophan residues highlighted in green.

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 10 below, which clearly shows an emission peak at 340 nm. Trp emits at shorter wavelengths (330-340 nm) when buried in a hydrophobic core of a protein [7] and at longer wavelengths (340-355 nm) when in a hydrophilic environment. The different Trp have different chemical surroundings, as expected from the varied distribution of 13 Trp residues in the structure model of 12R-LOX, as seen in figure 9, but the spectra clearly shows emission at 330-340 nm, hence indicating a folded protein.

Figure 10. Wavelength scan of intrinsic tryptophan fluorescence of 12R-LOX, showing a clear peak at 340 nm.

Nano Differential Scanning Fluorimetry

Foldedness and Thermal Stability

To further assess whether the enzyme was folded and to determine the thermal stability, nano Differential Scanning Fluorimetry (nanoDSF) was performed. As seen in figure 11 and figure 12, 12R-LOX has two unfolding transitions and hence we could determine two midpoints of thermal denaturation (Tm), as seen in table 1. 12R-LOX has two domains, and the two Tm indicate a correctly folded protein.

12R-LOX has 39 cysteine residues [3]. To study whether our enzyme had stabilizing disulfide bonds, we broke the bonds in our sample with dithiothreitol (DTT). The result can be seen in figure 11 and 12, showing that 12R-LOX appeared to be denatured before even thermal unfolding occurred, as there is no structure to unfold and therefore no cooperative unfolding events [8] to visualize with nanoDSF. These results further indicated that our enzyme is in its folded state, and with disulfide bonds.

Table 1. Tm for 12R-LOX in PBS buffer at pH 7.4.

Figure 11. Fluorescence intensity ratio between 350 nm/330 nm. Shows temperature when the enzyme begins to unfold. The graph is one of the replicates plotted and is representative of all the runs. The purple graph represents 12R-LOX alone, and the blue graph shows 12R-LOX in presence of DTT.

Figure 12. First derivative of the fluorescence intensity ratio between 350 nm/330 nm. The peaks show the TM for the two domains in 12R-LOX. The graph is one of the replicates plotted and is representative of all the runs. The purple graph represents 12R-LOX alone, and the blue graph shows 12R-LOX in presence of DTT.

Metal Co-Factors

To investigate the thermal stability in an environment more native to 12R-LOX, a different buffer, pH value and ions were present in the solution. It has been suggested that 12R-LOX possibly contain a Ca2+ binding domain and Fe2+ for its enzyme activity, as many other LOX proteins do [9]. Therefore, there was either Fe2+, Ca.2+ or both ten times excess the concentration of enzyme in the solution. As seen in figure 14, all experiments show a major unfolding event at 56.6 °C. This peak is similar to the stability of 12R-LOX in PBS buffer, as seen in figure 12, but the peak at 45.1°C has disappeared. Since this is the case in all runs in figure 13 and 14, this shows that the 10 mM Tris-HCl buffer and/or pH 7.0 stabilizes the enzyme. Instead, a minor unfolding transition at approximately 85°C occurs. The stability of 12R-LOX does not seem to be affected by Ca2+. However, for both experiments including Fe2+, there is an additional unfolding transition at 72.3°C, which suggests that Fe2+ has bound to the protein, which is not surprising since it is a cofactor for the enzyme activity of other LOX enzymes [9].

Table 2. Tm values for 12R-LOX when in presence of different ions and in 10 mM Tris-HCl.

Figure 13. Fluorescence intensity ratio between 350 nm/330 nm. Shows temperature when the enzyme begins to unfold. The graph is the average of the samples plotted and is representative of all the runs.

Figure 14. Fluorescence intensity of the first derivative of the ratio between 350 nm/330 nm. Shows temperature when the enzyme begins to unfold. The graph is the average of the samples plotted and is representative of all the runs. Visualized is 12R-LOX in 10 mM Tris-HCl buffer, pH 7.0. Either Fe2+, Ca2+ or both ions were present in the solutions to compare to 12R-LOX alone.

We decided to change the buffer to compare stability in presence of different ions and pH. The PBS buffer used in previous experiments had 0.14 M NaCl, 0.0027 M KCl and 0.01 M Phosphate and pH 7.4, and a higher pH value at 7.5. The higher pH was beneficial for the experiment with DTT, seen in figure 11 and 12, since the increased pH assists in breaking disulfide bonds. Additionally, the experiments with the Ca2+ metal ions cannot be performed in a phosphate buffer, since CaPO4 would precipitate. A lower pH at 7.0 was selected to prevent spontaneous chemical oxidation of Fe2+ to Fe3+ by molecular oxygen, O2, which involves reactive radical species.

Encapsulation in Liposomes

We encapsulated 12R-LOX in liposomes using thin film hydration and extrusion, the same way we encapsulated aeBlue (BBa_K864401) to obtain liposomes with a radius of 100 nm. After the enzyme was encapsulated, a SEC was performed to separate the liposomes from surrounding enzyme. The chromatogram from the SEC is shown in figure 15, and shows only one peak at UV 280 nm, where we expected two: one for the liposomes, and one for the surrounding enzyme. The fractions were eluted in the void colume for this column, and therefore the molecules have a Mw heavier than 2000 kDa, which is very heavy for a protein. To determine the protein and liposomes were not eluded in the same peak, even though it as very unlikely due to the Mw of the particles eluding in the void volume, we decided to add both aeBlue and 12R-LOX to the SEC. There was no elution for either protein, as seen in figure 15 and 16, and the column turned blue after 4x column volum had washed the column, as seen in figure 17, indicating that the column had high affinity for proteins, or at least for aeBlue.

Figure 15. The peak at 45.5 ml, i.e. the void volume, indicates the elution of liposomes, possibly with enzyme inside.

Figure 16. The peak at 44.2 ml, i.e. the void volume, indicates the elution of liposomes, possibly with enzyme inside.

Figure 17. The column after loading aeBlue and washing the column with multiple column volumes.

Microscopy

To investigate whether there was lipid structure in the sample, we used fluorescence microscopy with Nile red, which binds to liposomes and is visible during fluorescence microscopy, visualized in figure 18. Since Nile red has bound, this indicates lipid structure in the sample. The sample was also investigated using bright-field microscopy, which shows particles in visible light, seen in figure 19.

Figure 18. 12R-LOX with liposomes after SEC, in fluorescent light with excitation at 535 nm.

Figure 19. 12R-LOX with liposomes after SEC, in bright-field.

To compare, the samples for aeBlue, which is visible in visible light, resulted in the microscopy pictures seen in figure 20 and 21. Figure 21 is evidently different from figure 19 and the dots creating the contrasts are most likely the visible aeBlue inside the liposomes.

Figure 20. aeBlue with liposomes after SEC, in fluorescent light with excitation at 535 nm.

Figure 21. aeBlue with liposomes after SEC, in bright-field.

Dynamic Light Scattering

To assess the size of the lipid structures eluded fro the SEC, we performed a Dynamic Light Scattering (DLS) measurment. This showed that the size of the lipozomes was a radius of 100 nm for both samples, as seen in figure 22 and 23, indicating that we had intact liposomes.

Figure 22. To measure the size of the liposomes with 12R-LOX, a DLS was performed, showing particles with a radius of 100 nm.

Figure 23. To measure the size of the liposomes with aeBlue, a DLS was performed, showing particles with a radius of 100 nm.

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.

References

[1] Signor, L., Boeri Erba, E. Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa. J. Vis. Exp. (79), e50635, doi:10.3791/50635 (2013).

[2] Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Réfrégiers M, et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proceedings of the National Academy of Sciences [Internet]. 2015 Jun 2;112(24). Available from: https://doi.org/10.1073/pnas.1500851112

[3] UniProt [Internet]. Available from: https://www.uniprot.org/uniprotkb/O75342/entry

[4] Mihaly Varadi, Damian Bertoni, Paulyna Magana, Urmila Paramval, Ivanna Pidruchna, Malarvizhi Radhakrishnan, Maxim Tsenkov, Sreenath Nair, Milot Mirdita, Jingi Yeo, Oleg Kovalevskiy, Kathryn Tunyasuvunakool, Agata Laydon, Augustin Žídek, Hamish Tomlinson, Dhavanthi Hariharan, Josh Abrahamson, Tim Green, John Jumper, Ewan Birney, Martin Steinegger, Demis Hassabis, Sameer Velankar, AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences, Nucleic Acids Research, Volume 52, Issue D1, 5 January 2024, Pages D368–D375, https://doi.org/10.1093/nar/gkad1011

[5] Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). https://doi.org/10.1038/s41586-021-03819-2

[6] The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC

[7] Khrustalev Vladislav Victorovich, Khrustaleva Tatyana Aleksandrovna, Poboinev Victor Vitoldovich, Stojarov Aleksander Nicolaevich, Kordyukova Larisa Valentinovna, Akunevich Anastasia Aleksandrovna. Spectra of tryptophan fluorescence are the result of co-existence of certain most abundant stabilized excited state and certain most abundant destabilized excited state. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. Volume 257. 2021, 119784. ISSN 1386-1425. doi: 10.1016/j.saa.2021.119784.

[8] Whitford, David, and Inc Netlibrary. Proteins : Structure and Function. Hoboken, Nj, J. Wiley & Sons, 2005.

[9] Newcomer ME, Brash AR. The structural basis for specificity in lipoxygenase catalysis. Protein Sci. 2015 Mar;24(3):298-309. doi: 10.1002/pro.2626. Epub 2015 Jan 13. PMID: 25524168; PMCID: PMC4353356.

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