Part:BBa_K2929004:Experience

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iGEM 2019 St Andrews - Part Development

We had issues getting this part to express as a full-length protein in our E. coli BL21(DE3) chassis. Figure 1 shows a small-scale Ni purification of our mOrange+SpyTag product, with a band at ~15kDa in the eluted fraction. Mass spectrometry confirmed this band as being mOrange, despite the eluted fraction not being visibly Orange, and the band being at half the expected mass.

Expression of Yeast-Optimised mOrange (BBa_E2050) in our BL21(DE3) cells produced a visibly orange solution (figure 2), and a small-scale Ni purification of BBa_E2050 (figure 3) purified a band in the gel at the correct mass (~30kDa), and two other smaller bands. This suggested to us that the mOrange sequence is being cleaved post-translation, likely in a non-sequence-specific way, as mOrange is cleaved twice (at least) and mOrange+SpyTag at least once. This was also not an isolated issue, as replicates of both purifications returned the same result (see our wiki).

We attempted to assay the SpyTag capacity of the part by co-incubating the flow-through sample for our Ni purification of mOrange+SpyTag with an Open Isopeptide Domain (OIPD), equivalent to SpyCatcher. This was based on the rationale that the ~15kDa mass of N-6xHis tagged mOrange+SpyTag would have left approximately half the protein attached to a C-terminal SpyTag, which would be capable of ligating to OIPD. Unfortunately we observed no ligation for this (figure 4), but also observed no strong ~10kDa band in the flow-through (figure 1) indicative of expressed protein, so perhaps the protein is being cleaved twice (or more) and the bands become diffuse. If true, this cleavage could impede the ability of SpyTag to react with OIPD.

We suggest that teams wishing to characterise this part should analyse fluorescence of cultures expressing BBa_K2929004 in minimal media, to establish if this cleavage is occurring during the isolation process or if it occurs in the cell. Alternatively, teams may repeat our small-scale Ni purification protocol (see our wiki) but with Protease inhibitors in the isolation process to see if this has any effect.

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