Part:BBa_K2779900
HemA from R. capsulatus
This gene, which was codon-optimized for E. coli, encodes for 5-aminolevulinic acid synthase from Rhodobacter capsulitis. It catalyzes the formation of 5-aminolevulinate from succinyl CoA and glycine, according to the following scheme.
Characterization
Previous work has shown that overexpressing 5-aminolevulinic acid synthase can drive the expression of PPIX and that excess PPIX can be secreted in the media [1]. Thus, we transformed a plasmid carrying HemA under the pBAD promoter and cultured it in 250 mL of modified TB media supplemented with antibiotics and L-arabinose according to standard protocols. Using two slightly different protocols and solvent systems from previous work, we then collected the secreted porphyrins using DEAE-Sephadex A-25, an anion exchange resin. Elution of the adsorbed porphyrins from the resin allowed us to characterize the porphyrins via fluorescence and thin layer chromatography (TLC) and compare the results against that of a commercially available standard (referred to as “stock”).
The above figures show the normalized excitation (dotted) and emission (solid) spectra of the extracted porphyrins and the stock PPIX for both extraction solvent systems. While the literature emission peak [2] for PPIX (635 nm) differs from our confirmed stock’s observed peaks (608 nm and 666 nm), we believe that this is due to significant differences in solvent polarity as values reported in literature appear to be measured in aqueous solvents, and we have been measuring ours in an organic solvent-based system. A comparison of the spectra from the two extraction protocols and solvent systems suggest that the differences in the two solvent systems used (which differ by the addition of DMF and ratios of components) does not appear to have a significant effect on the observed peaks of the spectra. As the spectra show, there is a ~12 nm difference between the emission peaks of the stock PPIX and extracted porphyrins regardless of the solvent system used. While this discrepancy may be due to the extraction of a porphyrin species that is not PPIX, it is also just as likely to be due to a slightly different chemical environment in the extracted sample resulting from incomplete removal of the aqueous-based media, and/or calibration errors. Another observation that suggests the formation of PPIX is the similar shape of the extracted porphyrins’ spectrum to that of the stock solution, with the slight distortions being most likely caused by the inner filter effect due to a high sample concentration.
Our TLC protocols were adapted from a TLC solvent system defined by Kwon and coworkers [1]. As we did not have access to 1-chlorobutane, we omitted that component. The photo below show the TLC plates under UV light.
We tested the stock PPIX dissolved in each extraction solvent system (lanes 1 and 3 from the left on both plates) against the porphyrins extracted using both extraction solvent system (lanes 2 and 4 from the left on both plates). A comparison of the distance traveled by the spots reveals that there are two major species in each sample and in the case of the TLC, the extraction solvent affects the separation. While there are slight discrepancies in the distance travelled by the components of the extracted porphyrins when compared to the stock, we do not believe these discrepancies to be significant. Both extractions were performed on aliquots of the same culture, which means that they should have the same components. Yet, these results show that in one extraction solvent system, the extracted porphyrin travels a slightly smaller distance than the stock, while in the other system, the extracted porphyrin travels slightly larger distance. Taking the results of both comparisons into consideration, we are reasonably confident that the TLC supports the formation of PPIX. Indeed, the results of the TLC minimizes the possibility of our extracted porphyrins being intermediates or derivatives of PPIX since these compounds would have drastically different polarities due to differences in functional groups and their number. It is unlikely that the isolated porphyrin is protoporphyrinogen IX, the closest compound in terms of polarity to PPIX, because the oxidation of protoporphyrinogen IX to PPIX can be catalyzed by coporphyrinogen III oxidase in the presence of oxygen [1] and the culture was incubated in aerobic conditions. Lastly, each sample shows two major spots, indicating two major species present in each sample. Given that the second spot remains right at baseline, thus indicating very strong interactions with the polar silica, we believe that the two species in our sample are the protonated PPIX (compound that travelled up the plate) and the deprotonated PPIX.
References
[1] Kwon et al. Applied and environmental microbiology 2003, 69, 4875-4883.
[2] Hendrix Clinical chemistry 1983, 29, 1003-1003.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1219
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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