Part:BBa_J18932
mCherry RFP
Red fluorescent protein derived from DsRed.
Advantages:
- fast folding and maturation
- bright and photo-stable
Purity issues (update):
- Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
- Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)
- SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half (discussed in Gross et al. (2000) The structure of the chromophore within DsRed protein from coral.)
- Best maturation for expression at 20 or 25 C (rather than 37)
Usage and Biology
Characterization
Expression with BBa_K2319009
The protein was expressed under T7 promoter in E.coli BL21(DE3) with 6x-His tag at the N-terminal. The culture was induced at 37°C for three hours with a final IPTG concentration of 500μM. The cells were then lysed to obtain the protein. The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is its truncated counterpart.
Purification using Ni-NTA with BBa_K2319009
The cell lysate thus obtained was purified using Ni-NTA beads which only bind to proteins with a 6x-His tag, which is absent in the truncated protein. Ideally, the supernatant after binding should have the truncated protein and the elution after purification should have the non-truncated protein. This however is not true because the binding of 6xHis to Ni-NTA is not perfect.
Fluoroscence
Excitation Spectrum
The excitation spectrum of the purified sample (elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at 567 nm.
Emission Spectrum
The emission spectrum of the purified sample (elution) was obtained at a fixed excitation wavelength of 587 nm. The emission maxima was obtained at 605 nm
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.
Quantification of Truncation
The truncation of mCherry was determined by through two different methods:
- By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crude lysate.(rough estimation)
- By combining the fluorescence and gel intensity data of the Ni-NTA purification fractions (supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluorescence. The fluorescence of each of the above fractions was divided into fluorescence due to truncated and non-truncated protein based on their corresponding band intensities. The sum of fluorescence values of truncated and non-truncated protein were then used as a measure of their concentration to determine truncation.
Truncation Data
From gel intensity (rough estimation):
| % of protein | ||
| Truncated | Non-Truncated | |
| Replicate 1 | 30.25 | 69.75 |
| Replicate 2 | 48.6 | 51.4 |
| Replicate 3 | 35.8 | 64.2 |
| Replicate 4 | 34.2 | 65.8 |
| Replicate 5 | 49.8 | 50.2 |
| Average | 39.73 | 60.27 |
| Std.dev | 7.95 | 7.95 |
From this, mcherry is estimated to have a truncation of 39.73 % ± 7.95 %
From the calculations combining gel intensity and fluorescence:
| % of protein | ||
| Truncated | Non-Truncated | |
| Replicate 1 | 34.66 | 65.34 |
| Replicate 2 | 38.91 | 61.09 |
| Replicate 3 | 42.53 | 57.47 |
| Replicate 4 | 39.51 | 60.49 |
| Replicate 5 | 38.47 | 61.53 |
| Average | 38.82 | 61.18 |
| Std.dev | 2.52 | 2.53 |
From this, mcherry is estimated to have a truncation of 38.82 % ± 2.52 %
Improvement by IISc-Bangalore 2018
The sequence was modified to reduce the strength of the internal RBS and made into BBa_K2609006.Improvement by IISc-Bangalore 2019
The sequence was modified to reduce the truncation by replacement of the internal start codon with Isoleucine and made into BBa_K3165013.Improvement by NYCU-Taipei 2022
The sequence was modified by adding a tag and made into BBa_K4461013Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution: UniGE-Geneva
Group: UniGE-Geneva
Authors: Grégory Segala and Bryan Bourrat
Summary:In the list of Parts assigned to our team, we decided to characterize the fluorescent protein : mCherry RFP (BBa-J18932). Our goal was to observe the evolution of the intensity of fluorescence emitted by this protein, after induction by IPTG (Isopropyl -D-1-thiogalactopyranoside), varying two parameters: temperature and pH.
Documentation:
Conclusion: We can say that when the temperature increase, the fluorescence of mCherry decrease quite sharply. But, It is also observed that there is no variation in the intensity of fluorescence, depending on whether you are in an acidic, neutral or alkaline pH environment. Thus, mCherry seems less stable than the other fluorescent protein we characterized (sfGFP : BBa-I746916).
//proteindomain
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