Part:BBa_K3187008
GGGG-Tag for Sortase-mediated Ligation x mCherry Fluorescence Protein
Profile
| Name | GGGG-mCherry |
| Base pairs | 1028 |
| Molecular weight | 28.5 kDa |
| Origin | synthetic, derived from Discosoma sp. |
| Parts | mCherry, GGGG-sequence, T7 Promoter, lac Operator, GASPAG Linker, Strep-Tag II, T7 Terminator |
| Properties | Red fluorescent, Ex λ: 587nm, Em λ: 610 nm |
Usage and Biology
mCherry (BBa_K3187026) is a red
fluorescent
protein.
Which is a synthetic protein derived from Discosoma sp. by
directed evolution. The N-terminal GGGG-sequence (BBa_K3187018)
can be fused to a protein with a C-terminal LPETGG-Sortase A link (BBa_K3187019)
by Sortase A. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pET24 vector, containing the sequence of mCherry, a
GGGG-sequence, a GASPAG Linker
(BBa_K3187038), a
Strep-Tag II (BBa_K3187025)
for
Purification, a T7 Promoter with lac Operator and an RBS (BBa_K3187029), a T7 Terminator (BBa_K3187032), a Start-Codon (BBa_J70593)
and a Stop-Codon (BBa_K2868029). The
coding sequence consists of 851 bp which are translated to 260 amino acids.[1]
Results
FRET-based assay to analyze Sortase ligation efficiency
Results
Characterization of Sortase A7M (and comparison to Sortase A5M)
How do we measure if our purified sortases are active?
After purification of the sortases, we first performed SDS-PAGEs to verify that they are pure and monomeric. You can see in Fig. 3 that the purifications were successful. Next, we tested if the purified sortases connect two proteins that carry the important Sortase-recognition tags, N-terminal polyG and C-terminal LPETGG. Therefore, we added the sortases to a mix of GGGG-mCherry and mCherry-LPETGG. The reactions were performed in different buffers, at different enzyme-to-substrate ratios and for different time spans. We performed an SDS-PAGE, and prior to Coomassie staining, we recorded fluorescent images of the gel. Thereby, we could identify mCherry bands in the gel.
Figure x : SDS-PAGE of Sortase A7M and Sortase A5M where the bands show up at approximately 15 kDa. Our estimated size for Sortase A7M was 17.85 kDa, and for Sortase A5M 18.07 kDa. This confirms the result shown on the gel, since the band of Sortase A5M is a little higher than the one of Sortase A7M.
Figure x :
Fluorescence gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG
mediated by Sortase A7M incubated for 2 h and
4 h each. Reaction solutions were mixed with different ratios from enzyme to
substrate concentration(1:3;1:10) and each incubated in two different buffers(Tris-HCl
and Ammoniumdicarbonat).
Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from
left to right). The bands below the product at about 38 kDa could be semi-denatured
mCherry dimers.
b) Fluorescence gel on top of the coomassie-stained gel of the sortase-reaction
of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h
and
4 h each. Reaction solutions were mixed with different ratios from enzyme to
substrate concentration(1:3;1:10) and each incubated in two different buffers (Tris-HCl
and Ammoniumdicarbonat).
Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from
left to right). The bands below the product at about 38 kDa could be semi-denatured
mCherry dimers. Additionally, Sortase A7M can be seen at 17 kDaA7M.The
unprocessed mCherry monomers can be seen at 28 kDa.
As shown in Fig. 4, under certain conditions, a product band appeared at the expected size of 57.3 kDa (28.5+28.8 kDa). From this first activity test, we draw three conclusions:
- Our purified Sortase A7M is active
- The enzyme-substrate ratio affects the product yield
- The duration of the reaction affects the product yield
Additionally, TRIS buffer seems to alter the coomassie staining efficiency of Sortase A7M.
This endpoint measurement gave us a first impression that our Sortase A7M works nicely. Of
course, we wanted to further characterize the parameters of the reaction. When we understand
the Sortase better, modification of our VLPs will become more straightforward.
how do we measure sortase reaction kinetics
In the above described assays, we noticed the impact of enzyme-substrate ratio and reaction duration on the overall product yield. We thought about how to further measure the kinetics of the sortase reaction. In the literature, sortase reaction kinetics are often measured by FRET-assays. Therefore, we designed a suitable FRET-assay. In the end, we came up with a new FRET pair not described in the literature to date: 5-TAMRA-LPETG and GGGG-sfGFP.
Development of a new FRET pair
For characterization of the reaction kinetics of Sortase A7M, Sortase A5M and Sortase A, we decided to develop a suitable FRET pair. In order to find an optimal FRET pair, we first recorded an emission and absorption spectrum of 5-Carboxytetramethylrhodamin-LPETG (TAMRA) and GGGG-mCherry to verify the suitability for the FRET effect, checking for a possible overlap between the donor's emission and the acceptor's extinction.
Figure x : Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-mCherry (mCherry). In this configuration TAMRA acts as donor and mCherry as acceptor. When the two fluorophores are not linked via the substrates of the sortase only TAMRA is being excited. After sortase mediated ligation of the two substrates mCherry is the fluorophore being excited via the FRET and the emission of mCherry intensifies. Meanwhile, the emission of TAMRA decreases.
TAMRA is a chemical fluorophore that has an absorbance maximum at 542 nm and an emission
maximum at
570 nm. The
terminal carboxy
group of the dye was linked via a lysine linker to the LPETG sequence (see Fig. 5).
mCherry has
an N-terminal poly-glycine sequence and can therefore be linked to the LPETG motif of TAMRA via
the
Sortase A. For a sufficient FRET-effect, it is also necessary that the distance between
donor and
acceptor is lower than the Förster radius. The Förster radius describes the distance between two
fluorophores at which 50 % of the energy is transferred.
First, we wanted to identify which concentrations are needed for our experiment, then set up the
reaction
and measured fluorescence intensities. Over time, a decline in the emission of TAMRA can be
observed as
Sortase A7M/A5M is converting more educts to products.
Figure x : The graph shows the extinction and emission spectra of TAMRA and mCherry. Due to the large overlap of TAMRA emission and mCherry extinction it is possible to perform a FRET with this pair of fluorophores. The graph show the relative fluorescence unit (RFU[%]) in relation to the extincted/emitted wavelength [nm]. The peaks are normalized to 100 %.
The emission and extinction spectra of TAMRA and mCherry exhibit an overlap of emission of TAMRA and extinction of mCherry. Based on this output, a FRET-assay for the kinetics of Sortase A7M was performed to confirm whether the FRET-pair is working. As TAMRA is excited with light of a lower wavelength than mCherry, the former serves as FRET donor and the latter as acceptor. We chose the excitation wavelength at 485 nm to prevent unnecessary “leak” excitation of mCherry. Nevertheless, an extinction of mCherry could not be excluded and may have negative effects on the visibility of the FRET.
Figure x : Spectrum of TAMRA and mCherry, with Sortase A7M, over the course of 20 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.
The analysis of the data shown in Fig. 7 confirmed the aforementioned suspicion that mCherry is also excited at 485 nm, which makes differentiation of the fluorescence more difficult. Furthermore, Fig. 8 shows that the difference in the decline of TAMRA is not significant. Accordingly, a decline in the emission maximum of TAMRA over time is also visible in the negative control. One reason might be bleaching of TAMRA through the excitation by the laser. Nevertheless, conversion by the Sortase A7M can be observed by comparing the results with the negative control.
Figure x : Sortase reaction in TAMRA mCherry FRET after subtracting the negative control. Depicted is the difference in RFU over time [min]. WIthin the first 20 min of the substrate conversion is the quickest. At 30 min a plateau is reached. After 60 min starts catalyzing the reverse reaction. The mean ΔRFU value was normalized to zero for better visualization.
Comparison of MGGGG-mCherry and GGGG-mCherry (BBa_K2868010
References
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 854
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 854
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 915
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 854
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 854
- 1000COMPATIBLE WITH RFC[1000]
| None |