In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins
| Autor: | Hideo Iwaï, Edith Buchinger, Sara Züger, A. Sesilja Aranko |
|---|---|
| Přispěvatelé: | Institute of Biotechnology (-2009) |
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
dnaE
DNA polymerase Protein Conformation education Molecular Sequence Data lcsh:Medicine Peptide Biochemistry/Biocatalysis Protein Engineering 01 natural sciences Biochemistry Inteins src Homology Domains 03 medical and health sciences Protein structure Bacterial Proteins Protein splicing Biochemistry/Protein Chemistry Protein Splicing Chemistry/Biochemistry Amino Acid Sequence Biochemistry/Macromolecular Chemistry Biophysics/Biocatalysis lcsh:Science Nostoc Peptide sequence 030304 developmental biology DNA Polymerase III chemistry.chemical_classification 0303 health sciences Multidisciplinary biology 010405 organic chemistry lcsh:R Synechocystis Protein engineering 0104 chemical sciences chemistry biology.protein lcsh:Q Biotechnology/Protein Chemistry and Proteomics Ligation Research Article |
| Zdroj: | Aranko, A S, Züger, S, Buchinger, E & Iwai, H 2009, ' In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins ', PLOS ONE, vol. 4, no. 4, pp. e5185 . PLoS ONE PLoS ONE, Vol 4, Iss 4, p e5185 (2009) |
| Popis: | BACKGROUND: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. PRINCIPAL FINDINGS: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. CONCLUSION: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing. |
| Databáze: | OpenAIRE |
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