Difference between revisions of "Part:BBa K1362000"

Revision as of 18:09, 17 October 2014

NpuDnaE intein RFC[105] circularization construct

This part is an element of the [http://2014.igem.org/Team:Heidelberg Team Heidelberg 2014] intein toolbox and can be used as described in [LINK ZUM RFC|RFC[i]]. The purpose of this tool is to provide an easy way to circularize any linear protein. While conserving the functionality of their linear counterpart, circular proteins can be superior in terms of thermostability [1][2][3], resistance against chemical denaturation [4] and protection from exopeptidases [2][4]. Moreover, a circular backbone can improve in vivo stability of therapeutical proteins and peptides [5].

Using this part, BBa_K1362022 (Xylanase), BBa_K1362013 (lambda lysozyme) and DNA methyltransferase (DNMT1) were successfully circularized.

Protein Circularization by Split Inteins

Figure 2: Outline: split intein circularization

A well-reseached method to circularize large proteins is based on artificially split inteins. Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analogous to intron splicing on RNA level, this post translational modification was named intein splicing. The protein segments were called intein for internal protein sequence, and extein for external protein sequence, with upstream and downstram exteins termed N-exteins and C-exteins, respectively. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins, via a new peptide bond [E]. Some inteins like Npu DnaE are naturally split in two fragments, which can reassemble to to the active intein and thus ligate the exteins [F]. This process is called trans-splicing. If corresponding split inteins are added to both termini of a protein as illustrated in figure 2, the trans-splicing reaction results in a circular backbone.

Usage

Exteins, RFC [i] standard overhangs and BsaI sites have to be added to the coding sequence of the protein to be circularized without start- and stop codons by PCR. By Golden Gate assembly, the mRFP selection marker has to be replaced with the protein insert. After addition of an inducible promotor the circular protein is ready to be expressed. For detailed step-by-step instructions please use our Toolbox Guide.

Figure 3: In a single step the protein of interest is cloned into the split intein circularization construct (1.). The resulting plasmid (2.) can be used to express a fusion protein (3.). In an autocatalytic in vivo reaction, the circular protein (4.) is formed.

Results

Figure 4: Western Blot to prove circularization. In order to test whether lambda lysozyme is circular, we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.

Figure 5: Coomassie gel of circular xylanase and a linear control

Figure 6: The 3D structure of linear lambda lysozyme (purple) is compared with a model predicted by modeller (ref?) of circular lambda lysozyme (yellow) with a rigid linker (green)

Sequence and Features

References

[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638–13643 (1999).

[2] Iwai, H. & Plückthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166–72 (1999).

[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222–5235 (1956)

[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).

[5] Trabi, M. & Craik, D. J. Circular proteins--no end in sight. Trends Biochem. Sci. 27, 132–8 (2002).

[E] Perler, F. B. et al. Protein splicing elements: inteins and exteins — a definition of terms and recommended nomenclature. Nucleic Acids Res. 22, 1125–1127 (1994).

[F] Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–14 (2009).