Part:BBa_K2027000:Experience

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Applications of BBa_K2027000

To characterize the construct's (L-lysine Alpha Oxidase) function, we induced a reaction between the purified protein samples (samples in equilibration buffer, wash buffer, and elution buffer) with racemic lysine. H2O2 is a side product of the reaction between lysine and L-lysine alpha oxidase. Following the reaction induction, we performed the Amplex Red hydrogen peroxide assay on the protein purification fractions. We had a standard curve of H2O2 of 0-10 uM H2O2 (before the addition of Amplex Red Working Solution). Based on the plate data, the elution buffer fraction displayed the most H2O2 activity compared to the other fractions. This was plausible since the elution buffer fraction contains the greatest and most purified amount of L-lysine alpha oxidase.

Below is a screenshot of the previously mentioned plate data for our induced reaction and H2O2 assay with the enzyme. Wells A1 through A11 are part of the standard curve. Well A1-A10 are respectively 1-10 uM H2O2. Well A11 is 0 uM H2O2 and contains only the 1X reaction buffer that is part of the kit. Row B contains reactions that have been quenched after 30 minutes. Row C contains reactions that were induced 5 minutes before the plate was read by the spectrophotometer. Wells B1-B3 and C1-C3 contain the lysine reaction with varying amounts of elution buffer-protein solution, theoretically the purified enzyme eluted from the nickel column to which it was bound by a high concentration of the competitor imidazole. Wells B4-B9 and C4-C9 contain the lysine reaction with two different washes from the column; these may contain both weakly bound enzyme and residual soluble protein from the cell. Wells B10-B12 and C10-C12 contain the lysine reaction with varying amounts of equilibration wash buffer-protein solution, essentially all soluble protein that did not bind to the column during the incubation period.

We used the linear estimate derived from the standard curve below to analyze activity, tossing out row C (which we had hoped would have helped provide data on kinetics but in fact did not) and focusing on the elution and equilibration steps since both wash buffer samples and the elution buffer sample performed comparably on the assay.

Simply using the fact that the standard curve is more or less linear, a very rough approximation puts activity of the purified enzyme easily above twentyfold activity of the cell lysate incubated with the columns, indicating fairly strongly that the recombinant protein synthesized and tagged is effectively oxidizing L-lysine. Based on rough estimates, activity of our enzyme extracts was significantly lower than that reported by Tani et al. under similar conditions.¹ It is possible that this is due to an error in replication, possibly the performance of this reaction in closed tubes, and more study is needed. However, it is clear that this brick can lead to production of a functional purified product.

1. Tani, Y., Miyake, R., Yukami, R. et al. Appl Microbiol Biotechnol (2015) 99: 5045. doi:10.1007/s00253-014-6308-0

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