Difference between revisions of "Part:BBa K5301009"

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MCherry [11] is a part of bimolecular fluorescence complementation (BiFC) system.

MCherry [11] is a part of bimolecular fluorescence complementation (BiFC) system which could visualize protein interactions. The combination of mCherry [1-10] and mCherry [11] can form the fluorescence of mCherry. With its advantages of a short maturation time and brilliant fluorescence, the mCherry BiFC system could find particular applications for analyzing protein–protein interactions especially in living cells ^[1].

Usage and Biology

We devised the fusion expression of mCherry[11] with spNW15, SpyTag and SnoopCatcher (namely SnTST- mCh[11]). We used mCherry[1-10] and mCherry[11] parts to emit fluorescence to verify the success of protien connection. If SCSdC-mCh[1-10] can connect to SnTST- mCh[11] successfully, the mCherry[1-10] and mCherry[11] parts can emit fluorescence as a characterization.

We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1).

Characterization

We used Western Blot to test whether the protein containing mCherry[11] had been expressed successfully(Figure 2). The molecular weight of SnTST- mCh[11] (containing mCherry[11]) is 33.6 kDa. And we purified the target protein with a molecular weight of approximately 25-34 kDa (The red box), which demonstrated that we had successfully expressed the protein containing mCherry[11].

Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.

Sequence and Features

1. ↑ Fan, J., et al., Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochemical and Biophysical Research Communications, 2008. 367(1): p. 47-53.