Abstract
Our knowledge of biological systems relies increasingly on the ability of quantifying and imaging intracellular signals and events in living subjects. The development of novel methods and advances in biotechnology have provided many basic tools that allow analyses of the complex biological systems in living cells. Since the discovery of protein splicing in 1990, the elucidation of the splicing mechanism and the identification of key amino acid residues involved in the dissection and ligation of the peptide bonds have facilitated the molecular engineering of inteins for different applications in protein chemistry. These include protein purification, protein ligation and peptide cyclization, construction of split reporter proteins, regulation of protein activity, and introduction of non-natural amino acids. In this chapter, we focus on the construction of split reporter proteins and their applications for detecting protein- protein interactions, identification of organelle-localized proteins, growing safer transgenic plants, and screening antimycobacterial agents.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Bergelson J, Purrington CB, Wichmann G (1998) Promiscuity in transgenic plants. Nature 395:25
Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ (1997) Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 94:2306–2311
Buskirk AR, Ong YC, Gartner ZJ, Liu DR (2004) Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc Natl Acad Sci USA 101:10505–10510
Chin HG, Kim GD, Marin I, Mersha F, Evans TC Jr, Chen L, Xu M-Q, Pradhan S (2003) Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes. Proc Natl Acad Sci USA 100:4510–4515
Chong S, Xu MQ (1997) Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J Biol Chem 272:15587–15590
Conti E, Franks NP, Brick P (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4:287–298
David R, Richter MP, Beck-Sickinger AG (2004) Expressed protein ligation. Method and applications. Eur J Biochem 271:663–677
Davis EO, Jenner PJ, Brooks PC, Colston MJ, Sedgwick SG (1992) Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 71:201–210
Evans TJT, Xu M-Q (2002) Mechanistic and kinetic considerations of protein splicing. Chem Rev 102:4869–4884
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246
Gangopadhyay JP, Jiang SQ, Paulus H (2003) An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator. Anal Chem 75:2456–2462
Grimm S (2004) The art and design of genetic screens: mammalian culture cells. Nat Rev Genet 5:179–189
Hirata R, Ohsumi Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 265:6726–6733
Ho SN, Biggar SR, Spencer DM, Schreiber SL, Crabtree GR (1996) Dimeric ligands define a role for transcriptional activation domains in reinitiation. Nature 382:822–826
Kane PM, Yamashiro CT, Wolczyk DF, Neff N, Goebl M, Stevens TH (1990) Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science 250:651–657
Kawasaki M, Makino S, Matsuzawa H, Satow Y, Ohya Y, Anraku Y (1996) Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme. Biochem Biophys Res Commun 222:827–832
Kim SB, Ozawa T, Watanabe S, Umezawa Y (2004) High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase. Proc Natl Acad Sci USA 101:11542–11547
Lorenz WW, McCann RO, Longiaru M, Cormier MJ (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci USA 88:4438–4442
Martin DD, Xu M-Q, Evans TC Jr (2001) Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803. Biochemistry 40:1393–1402
Mootz HD, Muir TW (2002) Protein splicing triggered by a small molecule. J Am Chem Soc 124:9044–9045
Mootz HD, Blum ES, Tyszkiewicz AB, Muir TW (2003) Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 125:10561–10569
Nishiuchi Y, Inui T, Nishio H, Bodi J, Kimura T, Tsuji FI, Sakakibara S (1998) Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence. Proc Natl Acad Sci USA 95:13549–13554
Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395
Ozawa T, Umezawa Y (2002) Peptide assemblies in living cells. Methods for detecting protein-protein interactions. Supramol Chem 14:271–280
Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y (2000) A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal Chem 72:5151–5157
Ozawa T, Kaihara A, Sato M, Tachihara K, Umezawa Y (2001a) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem 73:2516–2521
Ozawa T, Nishitani K, Sako Y, Umezawa Y (2005) A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells. Nucleic Acids Res 33:e34
Ozawa T, Takeuchi TM, Kaihara A, Sato M, Umezawa Y (2001b) Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem 73:5866–5874
Ozawa T, Sako Y, Sato M, Kitamura T, Umezawa Y (2003) A genetic approach to identifying mitochondrial proteins. Nat Biotechnol 21:287–293
Paulmurugan R, Umezawa Y, Gambhir SS (2002) Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci USA 99:15608–15613
Paulus H (2003) Inteins as targets for potential antimycobacterial drugs. Front Biosci 8:s1157–s1165
Pietrokovski S (1998) Modular organization of inteins and C-terminal autocatalytic domains. Protein Sci 7:64–71
Shioda T, Andriole S, Yahata T, Isselbacher KJ (2000) A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening. Proc Natl Acad Sci USA 97:5220–5224
Westermann B, Neupert W (2003) ’Omics’ of the mitochondrion. Nat Biotechnol 21:239–240
Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 95:9226–9231
Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251
Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Ozawa, T., Umezawa, Y. (2005). Inteins for Split-Protein Reconstitutions and Their Applications. In: Belfort, M., Wood, D.W., Stoddard, B.L., Derbyshire, V. (eds) Homing Endonucleases and Inteins. Nucleic Acids and Molecular Biology, vol 16. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-29474-0_18
Download citation
DOI: https://doi.org/10.1007/3-540-29474-0_18
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-25106-4
Online ISBN: 978-3-540-29474-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)