Part:BBa_K1947025

BBa_K1947025

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 E. 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. 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.

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.

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.

Additionally, in order to know whether the Spycatcher, Spytag and blue pigment protein can bind together in prokaryotes and localized on the magnetic beads, we used a microscope to detect the blue pigment. As the result was shown in a blue pigment signal can be detected on the surface of magnetic beads.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 169
  Illegal NgoMIV site found at 253
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

This part serves as a catch system expressed in AMB-1.

References

Zakeri, B., et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. PNAS, 2012. 109(12): p. E690-7.