Team:TU Eindhoven/Contribution

Contribution

Contribution

Characterization mCherry

mCherry is a monomeric red fluorescent protein (mRFP) derived from DsRed of Discosoma sea anemones and is part of the mFruits family of mRFPs. DsRed is a tetrameric protein and has low photostability and high maturation time. mCherry was derived from the DsRed through directed evolution by Robert E. Campbell [1].

It absorbs light between 540-590 nm and emits light in the range of 550-650 nm. The gene consists of 711 base pairs and codes for 236 residues, resulting in a mass of 26.7 kDa. It contains three alpha-helices and thirteen beta-sheets making up a beta-barrel (Figure 1). The chromophore consists of Met66-Tyr67-Gly68. It has a lower molecular weight, improved brightness and higher photostability and folds faster than DsRed. It will, therefore, disturb the target system less and is widely used to visualize genes and analyze their functions and is of great significance in fluorescence microscopy as intracellular probe [2].




Figure 1: Crystal structure of mCherry [3].

While mCherry is used a lot, it is not yet highly characterized in the part registry. Therefore, we decided to add data of mCherry to the registry. First, mCherry (BBa_J06504) was cloned into a pET28a(+) vector (containing an N-terminal 6xHis-tag) and subsequently expressed in BL21 (DE3) E. coli and purified using immobilized metal affinity chromatography (IMAC). Different concentrations of mCherry were made and an increase in color intensity was visible (Figure 2). Consequently, the purified protein was analyzed on an SDS-PAGE (Figure 3) which shows a clear blob above 25 kDa, corresponding with 6xHis-tagged mCherry’s molecular weight of 29.3 kDa. The blob in between 10 kDa and 15 kDa is a truncated version of mCherry as described by Tripathi [4]. There are other bands visible so the purity is not optimal. However, the contrast between the huge blobs and the other bands is very big.




Figure 2: Purified mCherry in different concentrations, showing a clear increase in color intensity.



Figure 3: SDS-PAGE of mCherry after IMAC.

Fluorescence intensity measurement

Following protein purification, the protein functionality was analyzed. Firstly, the excitation and emission of mCherry were measured within its absorption and emission range (Figure 4). This shows a clear excitation maximum at 587 nm and an emission maximum at 610 nm. Secondly, the photostability of mCherry was measured following UV radiation up until thirty minutes to advance usage of mCherry in experiments (Figure 5). Exposure to UV light shows a decrease in fluorescence intensity. After 30 min, the intensity is almost halved compared to no UV exposure.


Figure 4: Excitation and emission spectrum of mCherry.


Figure 5: Excitation spectrum of mCherry after different periods of UV radiation.

References

  1. N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, “Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein,” Nat. Biotechnol., vol. 22, no. 12, pp. 1567–1572, 2004
  2. F. V Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods, vol. 6, no. 2, pp. 153–159, 2009.
  3. D. W. Piston, R. E. Campbell, R. N. Day, and M. W. Davidson, “Anthozoa Fluorescent Proteins.” [Online]. Available: http://zeiss-campus.magnet.fsu.edu/articles/probes/anthozoafps.html. [Accessed: 10-Oct-2019].
  4. R. Tripathi, “Functional characterisation of LEA proteins from bdelloid rotifers,” 2012.