Difference between revisions of "Part:BBa J70628"

Revision as of 15:30, 16 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 cleavage.

C-terminal cleavage is impossible because the required C-terminal 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 a Asn, Cys.

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]

@@ Line 4: / Line 4: @@
 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 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, it will remain bound to whatever matrix you used while your target protein will be washed through.  A typical assembly would be:
+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===
-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 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 cleavage.
+C-terminal cleavage is impossible because the required C-terminal 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 a Asn, Cys.
 <!-- -->