Part:BBa_K2144008
Coding sequence for Sortase A with His-tag
Usage
Sortase A is a bacterial enzyme with the ability to break and form new peptide bonds. The key feature of the enzyme is the specific conjugation reaction it carries out, where the enzyme recognizes a specific amino acid sequence, a so called sorting motif (LPXTG motif in the case of S.aureus) and conjugate this sequence with another unit carrying an oligo glycine motif where a new peptide bond is formed [1],[2].
Biology & BioBrick Design
This Biobrick is a truncated version of the enzyme where the transmembrane domain (amino acids 1-59) is not included in the coding sequence to increase solubility [2]. Further, the BioBrick has also been fused with the Protein G B1 Domain (GB1), upstream the Sortase coding sequence, acting as a solubility tag and a His-tag to enabling purification through IMAC [2].
Characterization by iGEM TU_Darmstadt 2019 (Expression and assays)
We purified the Sortase A from team Stockholm via fast protein liquid chromatography (FPLC) using the ĂKTA pure (fig. 2). In order to purify the Sortase A from team Stockholm we used the existing His-tag.
< ----- Bild von ĂKTA Chromatogram ---->Fig 1: Chromatogram of the ĂKTA during the purification of Sortase A from team Stockholm.
In figure 2 it is shown that the Sortase A from Stockholm eluted at ???. This should make sure that we have
purified the right protein with the fitting tag.
We then confirmed the data of the purification by checking on the size of the Sortase using SDS-PAGE (fig. 2).
We purified it twice with the same result in size.
Figure 2: A SDS-PAGE of a triplicate of the reaction solution for the sortase reaction consisting of coat protein (CP), mCherry and Sortase A from Stockholm which were incubated for 90 minutes at 37 °C. A negative control without Sortase A from Stockholm in a triplicate is also included. To verify the size the Sortase A from Stockholm was also put as a control on the gel solely.
In figure 2 a SDS-PAGE of the first sortase reaction performed with the Sortase A from Stockholm is shown as
well. On the right is a triplicate of the purified Sortase A from Stockholm to reassure it has the right size of
about 15 kDa which is the case. On the left are triplicates of coat protein with an LPETGG-tag (CP-LPETGG) and
GGGG-mCherry. The first three are with Sortase Stockholm in the reaction and the middle three are without
Sortase Stockholm. The reaction was run at 37 °C for 90 minutes. Seeing this first gel arose the suspicion that
the Sortase A from team Stockholm is not working properly. We then tried to confirm whether our suspicion was
right. In order to do so we ran another test with a SDS-PAGE (fig. 3) and additionally used the Sortase A from
Stockholm for one of our self-made FRET reactions.
Figure 3: A SDS-PAGE of the sortase reaction connecting CP-LPETGG and GGGG-mCherry. The gel includes a positive triplicate consisting of CP-LPETGG, GGGG-mCherry and Sortase A from Stockholm and two negative controls each in a triplicate of which one is similar to the positive control but without 10 mM calcium in the reaction buffer and one is without Sortase A in the reaction.
The gel in figure 3 shows a first proof for our suspicion. The first triplicate on the left contains CP-LPETGG,
GGGG-mCherry and Sortase from Stockholm in a buffer with 10 mM calcium present. The middle triplicate shows
CP-LPETGG, GGGG-mCherry and Sortase from Stockholm but in a buffer with no calcium present. The last triplicate
does only contain CP-LPETGG and GGGG-mCherry but no Sortase A from Stockholm. The reaction was performed at 37
°C for 90 minutes. As all three sample show the same outcome it can be assumend that the Sortase A from team
Stockholm does not show any activity whatsoever.
In order to have a final proof we compared our Sortase A7M to the Sortase A from Stockholm using a FRET
connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (fig. 4). We ran this
reaction for 3h at 30 °C.
Figure 4: A FRET-assay of Sortase A from Stockholm and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with and without 10mM calcium. The ÎRFU refers to the respective negeative control without Sortase.
As visible in figure 4 the Sortase A from Stockholm does not show any activity during the reaction although it
was incubated with 10mM calcium present. In contrary to our Sortase A7M which was incubated without calcium
because it is an independent mutant. The Sortase A7M also shows the expected increase in fluorescence visible in
the ÎRFU at 514 nm over time. The ÎRFU refers to the difference between the negative control without the
respective sortase (data not shown). In comparison the Stockholm Sortase A does not seem to catalyze any
reactions over the measured time span. If there would be any activity it would look similar to the graph of the
Sortase A7M where the ÎRFU at 514 nm is increased due to the FRET pair being connected. This confirms the
SDS-PAGEs that showed the same outcome of the Sortase A from Stockholm being not functional independent from the
presence of calcium.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Popp, M. W.-L. and Ploegh, H. L. (2011), Making and Breaking Peptide Bonds: Protein Engineering Using Sortase. Angew. Chem. Int. Ed., 50: 5024â503
[2] Westerlund, K. Karlstrom, A., Honarvar, H., Tolmachev, V. Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting. Bioconjugate Chem., 2015, 26 (8), pp 1724â1736
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