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 up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)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 fluoroscence.
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 crdue 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 intesity (rough esimation):
| % 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 fluorescense:
| % 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 strenght of the internal RBS and made into BBa_K2609006.Sequence 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]
//proteindomain
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