Difference between revisions of "Part:BBa K2144008"

Revision as of 09:39, 16 October 2019

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 FRET-based assay)

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Sequence and Features

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