![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K2926048
mCherry with hexahistidine tag for purification
This part codes for the red fluorescent protein mCherry with a C-terminal hexahistidine tag for simple purification via metal ions.
Usage and Biology
Because of the wide range of applications for fluorescing proteins there was a great interest in finding and engineering improved variants and a wider colour spectrum. In the last few years red fluorescing proteins became more and more important. Common native red fluorescing proteins are often dimeric or tetrameric what makes their usage in experimental setups difficult. Directed mutation of dsRFP from the corallimorpharia Discosoma sp. Led to the first monomeric red fluorescing protein mRFP1 (Shaner et al. 2004). Unfortunately this mutations resulted in a lower quantum yield and decreased photostability (Shaner et al. 2004). During further protein engineering attempts, scientists were able to create the red fluorescent protein mCherry. mCherry is a 26.7 kDa protein that shows a very short maturation time of about 15 minutes and a low acid sensitivity. Its excitation maximum lies at 587 nm and it has its emission maxiumum at 610 nm (www.fpbase.org). In 2006 the crystal structure of mCherry was published (Shu and Remington 2006).
![](https://2019.igem.org/wiki/images/7/7e/T--Bielefeld-CeBiTec--mCherry_Structure.png)
==Sequence and Features==
Sequence was validated by Sanger sequencing
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protein purification
The part mCherryHis was expressed, purified and characterized together with the parent part mCherry ( BBa_J06504 ).
To further characterize purified mCherry we compared two different purification protocols. Purification
via his-tag was compared to the IMPACT-purification protocol from NEB. For this purpose we cloned mCherry
( BBa_J06504 ) into the purification and expression vector pTXB1 from NEB and at the same time added six
histidines to the C-terminus of mCherry in pSB1C3 ( BBa_K2926048 ). Both expression vectors were transformed
in E. coli ER2566. After induction with IPTG both cultures showed the characteristic red colour of mCherry
expressing bacteria (Fig. 2 and Fig. 3).
![](https://2019.igem.org/wiki/images/3/30/T--Bielefeld-CeBiTec--mCherry_Cultures.png)
Expression cultures of mCherry in pTXB1 and mCherryHis in pSB1C3 in E. coli ER2566 were cultivated to an OD of around 0.6 at 37 °C in LB with 100 mg Ampicillin per L. Protein expression was induced by addition of IPTG to a final concentration of 0.4 mM. After additional 30 min at 37 °C cultures were transferred to 17 °C and protein expression was performed over night.
![](https://2019.igem.org/wiki/images/d/dc/T--Bielefeld-CeBiTec--mCherry_Harvested.png)
Expression cultures of mCherry and mCherryHis in E. coli ER2566 were harvested by centrifugation at 4 °C for 20 min and 4 000 rpm.
Purification
After cultivation and cell lysis via Ribolyzer the protein was purified using the His-purification kit from Macherey Nagel and the IMPACT-purification kit from NEB (Fig. 4).
![](https://2019.igem.org/wiki/images/a/ab/T--Bielefeld-CeBiTec--mCherry_purification-columns.png)
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 000 rpm for 15 s. The lysate was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm. Cleared lysate was loaded onto a chitin column (IMPACT-purification) or a Ni-TED column (purification via his tag) and washed with wash buffer. Finally the protein was eluted, washed in PBS and concentrated.
![](https://2019.igem.org/wiki/images/c/cf/T--Bielefeld-CeBiTec--mCherry_SDS-PAGE_MALDI.png)
E. coli lysate of the expression culture, flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98 °C for 10 min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12 %). The proteins were separated through electrophoresis (25 mA). Suggested mCherry bands in the lane with purified proteins were marked in dark red.
In the last lane you can see that we were able to purify mCherry as well as mCherryHis. While the IMPACT-purification resulted in a higher yield, the purity of mCherryHis was higher as the protein lane in Fig. 5 indicated.
Following the SDS-PAGE we analyzed the purified protein via MALDI-ToF. For this purpose we excised the marked bands (Fig. 5) from the SDS-PAGE and started a tryptic digestion of the washed gel fragment. Analysis via MALDI-ToF confirmed that we were able to purify mCherry (Fig. 6).
![](https://2019.igem.org/wiki/images/3/38/T--Bielefeld-CeBiTec--mCherry_MALDI.png)
Excised bands from the SDS-PAGE of mCherry and mCherryHis were washed, digested over night with trypsine and co-cristallyzed with a HCCA-matrix on a MALDI target. Mass spectrum was recorded in a MALDI-ToF MS from Bruker Daltronics and data was evaluated using the software BioTools.
Characterization
To gain some more knowledge about mCherry we analyzed different properties of the protein. First of all we measured its emission- and excitation spectra (Fig. 7).
![](https://2019.igem.org/wiki/images/0/06/T--Bielefeld-CeBiTec--mCherry_Spectra.png)
Emission- (dashed lines) and excitation-spectra (solid lines) of mCherry purified via IMPACT-Kit (dark purple) and His-tag (pink) were measured (λEx=570 nm, λEm=600 nm to 850 nm) using the TECAN infinite M200 and normalized to their maximum.
Next we compared the fluorescence intensity of the two different mCherry-variants normalized to Texas Red (Fig. 8).
![](https://2019.igem.org/wiki/images/a/a2/T--Bielefeld-CeBiTec--mCherry_Intensities.png)
Fluorescence intensity of a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured (λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red at the same wavelength.
References
Prasher, D. C.; Eckenrode, V. K.; Ward, W. W.; Prendergast, F. G.; Cormier, M. J. (1992): Primary structure of the Aequorea victoria green-fluorescent protein. In: Gene 111 (2).
Shaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y. (2004): Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. In: Nature biotechnology 22 (12).
Shu, X.; Remington, S. J. (2006): Crystal structure of mCherry.
Shu, Xiaokun; Shaner, Nathan C.; Yarbrough, Corinne A.; Tsien, Roger Y.; Remington, S. James (2006): Novel chromophores and buried charges control color in mFruits. In: Biochemistry 45 (32).