Part:BBa_K5301009

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 .

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).

Figure 1.The structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag as predicted by AlphaFold

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].

Figure 2.Western Blot analysis of the extraction results of SnTST- mCh[11](containing mCherry[11]).The molecular weight of SnTST- mCh[11] is 33.6kDa.

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.

Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]