Part:BBa_K2549043

CfaC-TEVpC

This part is one of the downstream elements of our amplifier. It is constructed by fusing CfaC (Part:BBa_K2549015) and TEVpC (Part:BBa_K2549010), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. TEVpC is the C-terminal fragment of TEV protease. TEV protease is one of the best-characterized enzymes of the viral proteases which have more stringent sequence specificity. When coexpressed with NLS-TEVpN-CfaN (Part:BBa_K2549042) in the same cell, two fusion proteins can form and the site-specific cleavage can be realized.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 91
  Illegal SapI.rc site found at 21
  

Biology

Please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement to check how we use this.

split TEV protease

MJ Rossner et al have demonstrated a biological assay termed split TEV. They engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins.

MJ Rossner et al demonstrated:In a systematic screen for transcomplementation-competent N and C-terminal TEV protease fragments (referred to as N-TEV and C-TEV), we found several fragment pairs that showed substantial recapitulation of activity upon specific interaction of model-protein dimerization domains to which they had been fused. We obtained the best results using the two fragment pairs N-TEV(1–118)/C-TEV(119–242) and N-TEV(1–70)/C-TEV(61–242). Both pairs displayed B30–40% activity compared to the full length TEV protease, a three- to fourfold increase over the control assay.We used the N-TEV(1–118)/C-TEV(119–242) pair for all subsequent experiments.

Thus in our split TEV protease assay, we chose site between 118 amino acid and 119 amino acid of TEV protease as the split site.

intein-based protein splicing

Schematic below provides a mechanism of how the intein function.

The mechanism of protein splicing involving inteins. In this scheme, the N-extein is shown in red, the intein in black, and the C-extein in blue. X represents either an oxygen or sulfur atom.

For more details about intein-based protein splicing, please refer to our CfaN (Part:BBa_K2549009) and CfaC (Part:BBa_K2549010).

a three-amino acids sequence added to the original TEVpC

Extein sequence preferences are largely confined to the catalytic cysteine at the +1 position and large hydrophobic residues that are preferred at the +2 position of the C-extein. The EKD to GEP mutation has drastically lowered the extein dependence of CfaC. Even so, we have added a Cys-Ala-Glu sequence to the original C-terminal fragment of the TEV protease to achieve higher efficiency of cleavage.

Steven AJ et al demonstrated:use of the more promiscuous CfaGEP split intein led to improved yields of cyclized product in all unfavorable +2 contexts. Furthermore, CfaGEP maintains this improved cyclization activity even when the −1 and +3 extein positions are varied.