Di Ventura Lab

Molecular and Cellular Engineering

Inteins are autocatalytic protein domains that excise themselves out of proteins and in doing so connect the flanking regions with a peptide bond. The splicing reaction requires the presence of specific amino acids that are conserved in all inteins, such as a cysteine or serine at the N-terminus and an asparagine at the C-terminus. Split inteins are constituted by two separate domains that need to associate to reconstitute a functional intein. Therefore, during the process of splicing, split inteins make fusions between two previously independent proteins or peptidic fragments. Inteins are extremely useful tools in synthetic biology, because they can be used to modify proteins of interest inside living cells. Split inteins can be for instance used to reconstitute a functional protein out of two dysfunctional split halves.

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Cartoon showing the concept of split intein-mediated fusion of two separate proteins.

In order for the splicing reaction to be efficient, some residues might have to be added to the protein of interest at the selected splice junction. These residues, called local exteins, depend on the intein family. Sometimes, artificial sequences are found to work even better than naturally selected ones, like in the case of the split Npu DnaE intein. The local exteins remain in the final product as a “scar”; thus, they might influence the activity of the protein after splicing. They should be selected with care, whenever it is not possible to splice a protein in a place where the appropriate residues are already present. Split inteins can be also used to circularize proteins. Circular proteins are proteins for which the N- and C-termini are linked by a peptide bond. These proteins typically are more thermostable and are resistant to the action of exopeptidases, properties that make them attractive for biotechnological applications. We have created two general-purpose circularization vectors based on two very efficient split inteins: Npu DnaE and gp41-1. Using these two plasmids we have circularized xylanase from Bacillus subtilis. Interestingly, we found that, when circularizing xylanase with only an additional serine, its melting temperature was increased of circa 7 degrees. Moreover, the protein became more tolerant to heat shock.

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Features and usage of the circularization vector pCIRCintein, where ‘intein’ stands for the selected split intein to be used. The insert is created by PCR amplification of the sequence coding for the protein of interest (POI). The sequences coding for the exteins and BsaI restriction sites have to be added by the primers. The insert is cloned in a one-pot reaction into pCIRC, replacing the mRFP expression cassette using Golden Gate assembly.

Currently we are using the split gp41-1 intein to control the activity of several important proteins in mammalian cells.

Publications

Backbone circularization of Bacillus subtilis family 11 xylanase increases its thermostability and its resistance against aggregation
M. C. Waldhauer, S. N. Schmitz, C. Ahlmann-Eltze, J. G. Gleixner, C. C. Schmelas, A. G. Huhn, C. Bunne, M. Buscher, M. Horn, N. Klughammer, J. Kreft, E. Schafer, P. A. Bayer, S. G. Kramer, J. Neugebauer, P. Wehler, M. P. Mayer, R. Eils, B. Di Ventura
Mol Biosyst, 2015
doi:10.1039/c5mb00341e