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)
At the start of our project, we looked for a Sortase A we could use for our project. We discovered a matching BioBrick in the registry that was submitted by the iGEM team Stockholm in the year 2016. We added an RBS upstream and a T7 terminator downstream of the coding sequence for the means of expression. We transformed our E. coli cells and started to express this Ca2+-dependent Sortase A. For comparison, we expressed Sortase A7M and Sortase A5M additional to Sortase A from Stockholm. As usual, we purified the Sortase A BBa_K2144008 via fast protein liquid chromatography (FPLC) using the ÄKTA pure (Fig. 1) by means of His-tag purification.
Figure 1: Chromatogram of the ÄKTA run. The elution of other proteins than Sortase A is shown.
Figure 2: Chromatogram of the ÄKTA run. Sortase A BBa_K2144008 eluted as a peak at about 59 mL.
In Fig.1 it is shown that the Sortase A BBa_K2144008 eluted at about 59 mL.Fig. 2 shows the elution of other proteins than the Sortase A. Expected size and purity of the protein were assessed by SDS-PAGE (Fig.3)
Figure 3: A SDS-PAGE with increased contrast 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 Fig.3 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 BBa_K2144008 to reassure it has the right size of about 15 kDa which is the case. The solvent front appears below the Sortase A BBa_K2144008 band. CP-LPETGG and GGGG-mCherry were mixed and Sortase A BBa_K2144008 was added. The reaction was run for 90 minutes at 37 °C. As a negative control, Sortase was omitted solely. The difference in the visibility of the bands of Sortase BBa_K2144008 in the positive and negative control resulted from the positive control being diluted 1:2 after the reaction. The other negative control includes CP-LPETGG and GGGG-mCHerry without the Sortase BBa_K2144008 added. As we expected to see a band at about 74 kDa in the positive control we suspected that the Sortase A BBa_K2144008 is not working properly. We then tried to confirm whether our suspicion was right by performing a FRET-assay.
In order to have a final proof we compared our Sortase A7M to the Sortase A BBa_K2144008 using a FRET-assay connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (Fig. 4). We measured the reaction kinetics 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 ΔRFU refers to the respective negative control without each sortase. .
As visible in Fig.4 the Sortase A BBa_K2144008 does not show any activity during the reaction although 10mM calcium was present in the reaction buffer. In contrary, the Sortase A7M, incubated without calcium, shows the expected increase in fluorescence visible in the ΔRFU at 514 nm over time. The ΔRFU refers to the difference between the negative control without the respective sortase. In comparison the Sortase A BBa_K2144008 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 ΔRFU at 514 nm is increased due to the FRET pair being connected. This confirms the SDS-PAGE results We have to conclude that Sortase A BBa_K2144008 is not functional.
- 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: 5024–503
[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 1724–1736
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