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 up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)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 <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>
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.
<img src="" width=100% style="border: 1px solid black;"> <figcaption>SDS PAGE with the cell lysate for WT uninduced, WT induced, K2319009 uninduced, K2319009 induced. The top band is the non-truncated protein and the the bottom band is the truncated protein.</figcaption></figure>
Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>
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.
<img src="" width=100% style="border: 1px solid black;"> <figcaption>SDS PAGE of fractions from Ni-NTA purification. The top band is the non-truncated protein and the bottom band is the protein truncated at the internal start codon (see arrowheads).</figcaption></figure>
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 576 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 607 nm
<figure style="width: 50%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em; clear:right;">
<img src="" width=100% style="border: 1px solid black;"><figcaption>Tht excitation and emission spectra of mCherry after normalizing it with WT BL21 (DE3) lysate.
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluoroscence. </figcaption> </figure>
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 after SDS PAGE.
- 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
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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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