Part:BBa_K1947025
BBa_K1947025 short
Usage and Biology
Protein purification is commonly used in Biochemical research, but can become a very tedious and inefficient procedure. We developed a system that can be used to conveniently and efficiently purify recombinant proteins with help of magnetosome. The system consists of two parts. First, we used Escherichia coli to express an interest protein tagged by a Spytag. Second, we generated a Spycatcher-fused Mms13 protein and expressed it in the magetotactic bacteria AMB-1 (Magnetospirillum magneticum). These bacteria synthesize magnetosomes covered by phospholipid bilayer membrane, in which Mms13 is tightly anchored. Spycatcher binds Spytag with high affinity, and thus Spycatcher-Mms13 anchored in magnetosomes can strongly bind Spytag-conjugated protein and specifically bring it down with help of magnetic field. Our system is applicable to efficiently purifying any interest protein for different research purposes.
There is a highly specific recognition and covalent conjugation between Spytag (a peptide with 13 amino acids) and Spycatcher (a small protein consisting of 138 residues). A protein of interest with a Spytag at its N- or C-terminus is expressed in E. coli and present in bacterial lysate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 169
Illegal NgoMIV site found at 253 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
This part serves as a catch system expressed in AMB-1.
Mms13 is a protein that bound to magnetosome directly and tightly on a bilayer phospholipid membrane of the bacterial magnetic particles (BMPs). GS linker is used to construct the fusion protein to prevent the two protein from influencing each other. And there is a highly specific recognition and covalent conjugation between Spytag (a peptide with 13 amino acids) and Spycatcher (a small protein consisting of 138 residues). The Spycatcher-Mms13-linked Magnetosome can specifically and covalently conjugate to the Spytag-tagged protein in the bacterial lysate and they can be simply co-purified by a magnet.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 169
Illegal NgoMIV site found at 253 - 1000COMPATIBLE WITH RFC[1000]
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