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RESEARCH - H. D. Mootz - Protein chemistry / biotechnology

Technology development for protein semisynthesis and protein functional control  

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The ability to site-selectively incorporate a synthetic moiety like a biophysical probe, an unnatural amino acid, or a defined posttranslational modification into a protein of interest opens up a variety of research avenues to interrogate protein structure and function that cannot be achieved by standard molecular biology approaches. Techniques for protein chemical modification have been around for a long time, however, new chemical reactions, sophisticated molecular biology approaches, and combinations thereof have taken protein chemistry to a new level in recent years.

We are interested in exploiting the power of the protein trans-splicing reaction catalyzed by split inteins for the selective chemical modification of proteins. Split intein fragments, intein-N and intein-C, link their fused sequences in a self-processing reaction with a native peptide bond and excise themselves during this process (see Figure 1)(1). They thus provide a means to link, in principle, any two polypeptide sequences, recombinant or synthetic (see Figures 2 and 3). The protein trans-splicing reaction offers several advantages over other approaches, like Native Chemical Ligation (NCL). For example, due to the high affinity of the intein fragments the reaction can be carried out at low reactant concentrations, it can be performed in complex mixtures like cell extracts or inside living cells, and no functional groups are required. However, it remains a challenge to identify inteins with better properties regarding the splicing efficiency, solubility, fragment sizes, and sequence tolerance for each particular application (1-4). A more detailed understanding of the protein splicing mechanism is also important to achieve these goals (5).

Since protein splicing involves a significant structural change of the fused peptide or protein sequences, an artificial conditional control on this reaction offers exciting opportunities to link to peptide or protein function. For example, we have designed a photocaged, synthetic intein fragment that can be used to trigger a split-intein mediated cleavage reaction by administration of UV-light. By releasing the active form of a coagulase in this way we were able allosterically induce thrombin activity in human blood plasma (6). In another approach we introduced a photocaged amino acid into a fully genetically encoded intein (Figure 4). With this system the protein splicing reaction and the formation of cyclic peptides could be turned on with light (7).

Selected references

  1. Mootz, H. D. (2009)
    Split inteins as versatile tools for protein semisynthesis, Chembiochem 10, 2579-2589.

  2. Zettler, J., Schütz, V., and Mootz, H. D. (2009)
    The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction, FEBS letters 583, 909-914.

  3. Thiel, I. V., Volkmann, G., Pietrokovski, S., and Mootz, H. D. (2014)
    An atypical naturally split intein engineered for highly efficient protein labeling, Angew Chem Int Ed 53, 1306-1310.

  4. Schütz, V., and Mootz, H. D. (2014)
    Click-tag and amine-tag: chemical tag approaches for efficient protein labeling in vitro and on live cells using the naturally split Npu DnaE intein, Angew Chem Int Ed 53, 4113-4117.

  5. Binschik, J., and Mootz, H. D. (2013)
    Chemical bypass of intein-catalyzed N-S acyl shift in protein splicing, Angew Chem Int Ed 52, 4260-4264.

  6. Binschik, J., Zettler, J., and Mootz, H. D. (2011)
    Photocontrol of protein activity mediated by the cleavage reaction of a split intein, Angew Chem Int Ed 50, 3249-3252.

  7. Böcker, J. K., Friedel, K., Matern, J. C., Bachmann, A. L., and Mootz, H. D. (2015)
    Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides, Angew Chem Int Ed 54, 2116-2120.

  8. Ludwig, C., Pfeiff, M., Linne, U., and Mootz, H. D. (2006)
    Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing, Angew Chem Int Ed 45, 5218-5221.

  9. Ludwig, C., Schwarzer, D., and Mootz, H. D. (2008)
    Interaction studies and alanine scanning analysis of a semi-synthetic split intein reveal thiazoline ring formation from an intermediate of the protein splicing reaction, J Biol Chem 283, 25264-25272.

  10. Kurpiers, T., and Mootz, H. D. (2007)
    Regioselective cysteine bioconjugation by appending a labeled cystein tag to a protein by using protein splicing in trans, Angew Chem Int Ed 46, 5234-5237.

Last update: 2015/11

Technology development for
protein semisynthesis
Posttranslational modifications
of the Ubiquitin-type
Coordination in multi-domain
biosynthesis enzymes

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