Part:BBa_K3755004

cfa-N

Codon optimized N-terminal spontaneous splicing−ligation peptide sequnece for 3*mfp5 CDS assembly, connected downstream of the 2*mfp5 CDS.

Usage and Biology

Split inteins(SIs) are autocatalytic peptides that catalyze the spontaneous splicing−ligation reactions between two SI-fused target proteins, assembling the two target proteins covalently in trans^[1]. A recently engineered Cfa SI was employed due to its rapid protein splicing rate in denaturing conditions (e.g., 8 M urea), which we used to efficiently extract and solubilize the target Mfp5 proteins^[2].

The Cfa-n is the N-terminal Cfa SI which is genetically fused to 2*Mfp5 CDS when Cfa-c is fused to 1*Mfp5 CDS. The two fusion proteins in whole cell lysate mixtures were then mixed to yield 3 repeats of Mfp5 protein, which can not be expressed by simple 3*Mfp5 CDS^[3].

We learned form Eugene Kim's literatur that repeating the sequence of Mfp5 can lead to higher adhesion^[3]. So, we built the expression system of 3 repeats of Mfp5 protein(Mfp5-3) to improve our Mfp5-1(part:BBa_K3755011) and Mfp5-2(part:BBa_K3755012). The expression system consists of two composite part part:BBa_K3755029 and part:BBa_K3755030, and this part is the basic component of part:BBa_K3755030.

Experiment and Results

We characterized this part in a larger composite part part:BBa_K3755030, please go to that page for more details.

References

[1]Carvajal-Vallejos P, Pallissé R, Mootz HD, Schmidt SR. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem. 2012 Aug 17;287(34):28686-96. doi: 10.1074/jbc.M112.372680. Epub 2012 Jun 28. PMID: 22753413; PMCID: PMC3436554.

[2]Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW. Design of a Split Intein with Exceptional Protein Splicing Activity. J Am Chem Soc. 2016 Feb 24;138(7):2162-5. doi: 10.1021/jacs.5b13528. Epub 2016 Feb 8. PMID: 26854538; PMCID: PMC4894280.

[3]Kim E, Dai B, Qiao JB, Li W, Fortner JD, Zhang F. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces. 2018 Dec 12;10(49):43003-43012. doi: 10.1021/acsami.8b14890. Epub 2018 Nov 27. PMID: 30480422. 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
  COMPATIBLE WITH RFC[1000]