Abstract
Inteins are protein segments embedded in frame within a precursor sequence that catalyze a self-excision reaction and ligate the flanking sequences with a standard peptide bond. Split inteins are expressed as two separate polypeptide fragments and trans-splice upon subunit association. Split inteins have found use in biotechnology applications but their use in postsynthetic domain assembly in vivo has been limited to the ligation of two protein domains. Alternatively, they have been used to splice three domains and fragments in vitro. To further develop split intein-based applications in vivo, we have designed a cell-based assay for the postsynthetic splicing of three protein domains using orthogonal split inteins. Using naturally and artificially split inteins, NpuDnaE and SspDnaB, we show that a multidomain protein of 128 kDa can be assembled in Escherichia coli from individually expressed domains. In the current system, the main bottleneck in achieving high yield of tandem trans-spliced product appears to be the limited solubility of the SspDnaB precursors. Optimizing protein solubility should be important to achieve efficient combinatorial synthesis of protein domains in the cell.
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References
Shah, N. H., & Muir, T. W. (2014). Inteins: nature’s gift to protein chemists. Chemical Science, 5, 446–461.
Topilina, N. I. and Mills, K. V. (2014). Recent advances in in vivo applications of intein-mediated protein splicing. Mobile DNA, 5, 5
Southworth, M. W., Amaya, K., Evans, T. C., Xu, M. Q. and Perler, F. B (1999). Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques, 27, 110–114, 116, 118–120
Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol, 17, 889–892.
Wu, W. Y., Mee, C., Califano, F., Banki, R., & Wood, D. W. (2006). Recombinant protein purification by self-cleaving aggregation tag. Nat Protoc, 1, 2257–2262.
Lew, B. M., Mills, K. V., & Paulus, H. (1999). Characteristics of protein splicing in trans mediated by a semisynthetic split intein. Biopolymers, 51, 355–362.
Perler, F. B. (2002). InBase: the Intein Database. Nucleic Acids Res, 30, 383–384.
Giriat, I., & Muir, T. W. (2003). Protein semi-synthesis in living cells. J Am Chem Soc, 125, 7180–7181.
Kwon, Y., Coleman, M. A., & Camarero, J. A. (2006). Selective immobilization of proteins onto solid supports through split-intein-mediated protein trans-splicing. Angew Chem Int Ed Engl, 45, 1726–1729.
Wong, S. S., Kotera, I., Mills, E., Suzuki, H., & Truong, K. (2012). Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators. Cell Calcium, 51, 57–64.
Ramirez, M., Guan, D., Ugaz, V., & Chen, Z. (2013). Intein-triggered artificial protein hydrogels that support the immobilization of bioactive proteins. J Am Chem Soc, 135, 5290–5293.
Guan, D., Ramirez, M., & Chen, Z. (2013). Split intein mediated ultra-rapid purification of tagless protein (SIRP). Biotechnol and Bioeng, 110, 2471–2481.
Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C., & Benkovic, S. J. (1999). Production of cyclic peptides and proteins in vivo. Proc Natl Acad Sci U S A, 96, 13,638–13,643.
Bionda, N., Cryan, A. L., & Fasan, R. (2014). Bioinspired strategy for the ribosomal synthesis of thioether-bridged macrocyclic peptides in bacteria. ACS Chem Biol, 9, 2008–2013.
Evans Jr., T. C., Benner, J., & Xu, M. Q. (1999). The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum. J Biol Chem, 274, 3923–3926.
Mootz, H. D. (2009). Split inteins as versatile tools for protein semisynthesis. Chembiochem, 10, 2579–2589.
Muralidharan, V., & Muir, T. W. (2006). Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Meth, 3, 429–438.
Yamazaki, T., Otomo, T., Oda, N., Kyogoku, Y., Uegaki, K., Ito, N., Ishino, Y., & Nakamura, H. (1998). Segmental Isotope Labeling for Protein NMR Using Peptide Splicing. J Am Chem Soc, 120, 5591–5592.
Muona, M., Aranko, A. S., Raulinaitis, V., & Iwai, H. (2010). Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro. Nat Protoc, 5, 574–587.
Shi, J., & Muir, T. W. (2005). Development of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split Inteins. J Am Chem Soc, 127, 6198–6206.
Shah, N. H., Vila-Perello, M., & Muir, T. W. (2011). Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein. Angewandte Chemie (International ed. in English), 50, 6511–6515.
Aranko, A. S., Oeemig, J. S., Zhou, D., Kajander, T., Wlodawer, A., & Iwai, H. (2014). Structure-based engineering and comparison of novel split inteins for protein ligation. Mol Biosyst, 10, 1023–1034.
Zuger, S., & Iwai, H. (2005). Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nat Biotech, 23, 736–740.
Brenzel, S., Kurpiers, T., & Mootz, H. D. (2006). Engineering artificially split inteins for applications in protein chemistry: biochemical characterization of the split Ssp DnaB intein and comparison to the split Sce VMA intein. Biochemistry, 45, 1571–1578.
Iwai, H., Zuger, S., Jin, J., & Tam, P. H. (2006). Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett, 580, 1853–1858.
Zettler, J., Schutz, V., & Mootz, H. D. (2009). The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett, 583, 909–914.
Wu, H., Xu, M. Q., & Liu, X. Q. (1998). Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein. Biochim Biophys Acta, 1387, 422–432.
Sun., W., Yang, J. and Liu, X. Q. (2004). Synthetic two-piece and three-piece split inteins for protein trans-splicing. J Biol Chem, 279, 35,281–35,286
Wu, H., Hu, Z., & Liu, X. Q. (1998). Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A, 95, 9226–9231.
Caspi, J., Amitai, G., Belenkiy, O., & Pietrokovski, S. (2003). Distribution of split DnaE inteins in cyanobacteria. Mol Microbiol, 50, 1569–1577.
Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z., & Muir, T. W. (2012). Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein Engineering. J Am Chem Soc, 134, 11,338–11,341.
Demonte, D., Dundas, C. M., & Park, S. (2014). Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl Microbiol Biotechnol, 98, 6285–6295.
Lim, K. H., Huang, H., Pralle, A., & Park, S. (2013). Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol Bioeng, 110, 57–67.
Cheriyan, M., Pedamallu, C. S., Tori, K., & Perler, F. (2013). Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. J Biol Chem, 288, 6202–6211.
Shah, N. H., Eryilmaz, E., Cowburn, D., & Muir, T. W. (2013). Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing. J Am Chem Soc, 135, 5839–5847.
Cane, D. E., Walsh, C. T., & Khosla, C. (1998). Harnessing the biosynthetic code: combinations, permutations, and mutations. Science, 282, 63–68.
Baltz, R. H. (2006). Molecular engineering approaches to peptide, polyketide and other antibiotics. Nat Biotechnol, 24, 1533–1540.
Fischbach, M. A., Lai, J. R., Roche, E. D., Walsh, C. T., & Liu, D. R. (2007). Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Proc Natl Acad Sci U S A, 104, 11,951–11,956.
Evans, B. S., Chen, Y., Metcalf, W. W., Zhao, H., & Kelleher, N. L. (2011). Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo. Chem Biol, 18, 601–607.
Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison 3rd, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Meth, 6, 343–345.
Engler, C., Kandzia, R. and Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. PLoS One, 3, e3647
Carvajal-Vallejos, P., Pallisse, R., Mootz, H. D., & Schmidt, S. R. (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem, 287, 28,686–28,696.
Aranko, A. S., Wlodawer, A., & Iwai, H. (2014). Nature’s recipe for splitting inteins. Protein Eng Des Sel, 27, 263–271.
Smialowski, P., Martin-Galiano, A. J., Mikolajka, A., Girschick, T., Holak, T. A., & Frishman, D. (2007). Protein solubility: sequence based prediction and experimental verification. Bioinformatics, 23, 2536–2542.
Huang, H. L., Charoenkwan, P., Kao, T. F., Lee, H. C., Chang, F. L., Huang, W. L., Ho, S. J., Shu, L. S., Chen, W. L. and Ho, S. Y. (2012). Prediction and analysis of protein solubility using a novel scoring card method with dipeptide composition. BMC Bioinformatics, 13 Suppl 17, S3
Selgrade, D. F., Lohmueller, J. J., Lienert, F., & Silver, P. A. (2013). Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells. J Am Chem Soc, 135, 7713–7719.
Thompson, K. E., Bashor, C. J., Lim, W. A., & Keating, A. E. (2012). SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth Biol, 1, 118–129.
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This research was supported by a grant from the National Science Foundation (grant No. 1264051).
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Demonte, D., Li, N. & Park, S. Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins. Appl Biochem Biotechnol 177, 1137–1151 (2015). https://doi.org/10.1007/s12010-015-1802-0
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DOI: https://doi.org/10.1007/s12010-015-1802-0