Difference between revisions of "Part:BBa K1947025"
 
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<h2>BBa_K1947025 short</h2>
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<h2>BBa_K1947025</h2>
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<h3>Usage and Biology</h3>
 
<h3>Usage and Biology</h3>
  
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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.
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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 <i>E. coli</i> 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.  
  
  
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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.
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[[File:T--Official_NEFU_China--File_whole_.png|800px|thumb|center]]
  
  
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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.
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K1947025 SequenceAndFeatures</partinfo>
 
  
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[[File:T--Official_NEFU_China--File_252_ (2).png|500px|thumb|center]]
  
  
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===Functional Parameters===
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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 <i>E. coli</i> and present in bacterial lysate.
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<partinfo>BBa_K1947025 parameters</partinfo>
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__NOTOC__
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<partinfo>BBa_K1947025 short</partinfo>
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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.  
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[[File:T--Official_NEFU_China--File_253_ (2).png|500px|thumb|center]]
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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.
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[[File:T--Official_NEFU_China--File_254_ (3).png|500px|thumb|center]]
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[[File:T--Official_NEFU_China--File_255_ (2).png|500px|thumb|center]]
  
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1947025 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1947025 SequenceAndFeatures</partinfo>
  
  
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1947025 parameters</partinfo>
 
<partinfo>BBa_K1947025 parameters</partinfo>
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<!-- -->
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__NOTOC__
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<partinfo>BBa_K1947025 short</partinfo>
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===References===
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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.

Latest revision as of 02:52, 30 October 2016

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.


T--Official NEFU China--File whole .png


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.


T--Official NEFU China--File 252 (2).png


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.

T--Official NEFU China--File 253 (2).png


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.


T--Official NEFU China--File 254 (3).png


T--Official NEFU China--File 255 (2).png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 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.