Difference between revisions of "Part:BBa J70628"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_J70628 short</partinfo>
 
<partinfo>BBa_J70628 short</partinfo>
−
This is a RFC12 compatible Intein self-splicing protein.  It is capable of self cleavage induced by overnight incubation at 4°C with 50 mM DDT.
 
  
 +
This is a RFC12 compatible Intein self-splicing protein.  It is capable of self cleavage induced by overnight incubation at 4°C with 50 mM DDT.
 +
 +
Note that this is an internal domain meant for '''only N-terminal fusions'''. This particular Intein has been designed by NEB such that the C-terminus will not be cleaved, which means that you can assemble this part with a given affinity tag (such as <partinfo>J70626</partinfo>), and then any protein assembled upstream of this part can be purified via the affinity tag and separated from the tag by this intein. Because the intein cannot cleave at its C-terminus, the intein and tag will remain bound to whatever matrix you used while your target protein will be washed through.  A typical assembly would be:
 +
 +
Target_Protein + Intein - CBD
 +
 +
which will only be cleaved at the + link.
 
===Usage and Biology===
 
===Usage and Biology===
−
Intein domains are commonly fused with CBD (<partinfo>J70627</partinfo>) and then used to cleave off the CBD tag after purification, yielding very high levels of the target protein. Note that the target protein's N terminal or C terminal residue can affect cleavage.
+
Intein domains are commonly fused with CBD (<partinfo>J70627</partinfo>) and then used to cleave off the CBD tag after purification, yielding very high levels of the target protein. Note that the target protein's C-terminal residue can affect the intein's N-terminal cleavage.
 +
 
 +
C-terminal cleavage is impossible because the required C-terminal splice junction Asparagine,Cysteine 545,546 residues have been changed to an Alanine.  The last three translated codons are gtc, cat, gca ; these codons translate to Val, His, Ala.  If you wish the protein to cleave the C-terminal correctly, those three must be replaced with Val, His, Asn, Cys.  Essentially the Alanine must be changed to an Asn, Cys.  Note that those four residues do not have to be the last four, they just have to be correct so that the intein can splice at that site.
 +
 
 +
Remember, however, that if you splice at both junctions then your target protein will not be the only thing that washes through the matrix. 
  
 
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Latest revision as of 14:31, 19 August 2010

RFC12 Sce VMA Intein Internal Domain

This is a RFC12 compatible Intein self-splicing protein. It is capable of self cleavage induced by overnight incubation at 4°C with 50 mM DDT.

Note that this is an internal domain meant for only N-terminal fusions. This particular Intein has been designed by NEB such that the C-terminus will not be cleaved, which means that you can assemble this part with a given affinity tag (such as BBa_J70626), and then any protein assembled upstream of this part can be purified via the affinity tag and separated from the tag by this intein. Because the intein cannot cleave at its C-terminus, the intein and tag will remain bound to whatever matrix you used while your target protein will be washed through. A typical assembly would be:

Target_Protein + Intein - CBD

which will only be cleaved at the + link.

Usage and Biology

Intein domains are commonly fused with CBD (BBa_J70627) and then used to cleave off the CBD tag after purification, yielding very high levels of the target protein. Note that the target protein's C-terminal residue can affect the intein's N-terminal cleavage.

C-terminal cleavage is impossible because the required C-terminal splice junction Asparagine,Cysteine 545,546 residues have been changed to an Alanine. The last three translated codons are gtc, cat, gca ; these codons translate to Val, His, Ala. If you wish the protein to cleave the C-terminal correctly, those three must be replaced with Val, His, Asn, Cys. Essentially the Alanine must be changed to an Asn, Cys. Note that those four residues do not have to be the last four, they just have to be correct so that the intein can splice at that site.

Remember, however, that if you splice at both junctions then your target protein will not be the only thing that washes through the matrix.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 486
    Illegal BamHI site found at 1339
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1195
  • 1000
    COMPATIBLE WITH RFC[1000]