Segmental Isotopic Labeling of Multi-domain and Fusion Proteins by Protein Trans-splicing in Vivo and in Vitro

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2010 Mar 6

PMID 20203672

Citations 34

Authors

Mikko Muona

A Sesilja Aranko

Vytas Raulinaitis

Hideo Iwai

Affiliations

Soon will be listed here.

Abstract

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.

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References

EVANS Jr T, Benner J, Xu M . Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 1998; 7(11):2256-64. PMC: 2143866. DOI: 10.1002/pro.5560071103. View

Paulus H . Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem. 2000; 69:447-96. DOI: 10.1146/annurev.biochem.69.1.447. View

Han J, Han G . A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal Biochem. 1994; 220(1):5-10. DOI: 10.1006/abio.1994.1290. View

Zuger S, Iwai H . Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nat Biotechnol. 2005; 23(6):736-40. DOI: 10.1038/nbt1097. View

Getz E, Xiao M, Chakrabarty T, Cooke R, Selvin P . A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem. 1999; 273(1):73-80. DOI: 10.1006/abio.1999.4203. View

Zhou P, Lugovskoy A, Wagner G . A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. J Biomol NMR. 2001; 20(1):11-4. DOI: 10.1023/a:1011258906244. View

Zhang X, Studier F . Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol. 1997; 269(1):10-27. DOI: 10.1006/jmbi.1997.1016. View

Studier F . Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991; 219(1):37-44. DOI: 10.1016/0022-2836(91)90855-z. View

Zettler J, Schutz V, Mootz H . The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 2009; 583(5):909-14. DOI: 10.1016/j.febslet.2009.02.003. View

10.

Nallamsetty S, Waugh D . Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr Purif. 2005; 45(1):175-82. DOI: 10.1016/j.pep.2005.06.012. View

11.

Yagi H, Tsujimoto T, Yamazaki T, Yoshida M, Akutsu H . Conformational change of H+-ATPase beta monomer revealed on segmental isotope labeling NMR spectroscopy. J Am Chem Soc. 2004; 126(50):16632-8. DOI: 10.1021/ja045279o. View

12.

Skrisovska L, Allain F . Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L. J Mol Biol. 2007; 375(1):151-64. DOI: 10.1016/j.jmb.2007.09.030. View

13.

Aranko A, Zuger S, Buchinger E, Iwai H . In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. PLoS One. 2009; 4(4):e5185. PMC: 2664965. DOI: 10.1371/journal.pone.0005185. View

14.

Busche A, Aranko A, Talebzadeh-Farooji M, Bernhard F, Dotsch V, Iwai H . Segmental isotopic labeling of a central domain in a multidomain protein by protein trans-splicing using only one robust DnaE intein. Angew Chem Int Ed Engl. 2009; 48(33):6128-31. DOI: 10.1002/anie.200901488. View

15.

Wider G, Wuthrich K . NMR spectroscopy of large molecules and multimolecular assemblies in solution. Curr Opin Struct Biol. 1999; 9(5):594-601. DOI: 10.1016/s0959-440x(99)00011-1. View

16.

Dillon P, Rosen C . A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques. 1990; 9(3):298, 300. View

17.

Serber Z, Dotsch V . In-cell NMR spectroscopy. Biochemistry. 2001; 40(48):14317-23. DOI: 10.1021/bi011751w. View

18.

Oeemig J, Aranko A, Djupsjobacka J, Heinamaki K, Iwai H . Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett. 2009; 583(9):1451-6. DOI: 10.1016/j.febslet.2009.03.058. View

19.

Brinkmann U, Mattes R, Buckel P . High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene. 1989; 85(1):109-14. DOI: 10.1016/0378-1119(89)90470-8. View

20.

Aachmann F, Svanem B, Guntert P, Petersen S, Valla S, Wimmer R . NMR structure of the R-module: a parallel beta-roll subunit from an Azotobacter vinelandii mannuronan C-5 epimerase. J Biol Chem. 2006; 281(11):7350-6. DOI: 10.1074/jbc.M510069200. View