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http://dx.doi.org/10.4014/jmb.0908.08013

Protein Cyclization Enhanced Thermostability and Exopeptidase-Resistance of Green Fluorescent Protein  

Zhao, Zhonglin (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Ma, Xin (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Li, Liang (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Zhang, Wei (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Ping, Shuzhen (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Xu, Ming-Qun (New England Biolabs, Inc.)
Lin, Min (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Publication Information
Journal of Microbiology and Biotechnology / v.20, no.3, 2010 , pp. 460-466 More about this Journal
Abstract
A mutant of green fluorescent protein ($GFPmut3^*$) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of $GFPmut3^*$ was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear $GFPmut3^*$. The circular $GFPmut3^*$ was $5^{\circ}C$ more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular $GFPmut3^*$ also displayed increased relative fluorescence intensity. In addition, chemical stability of $GFPmut3^*$ against GdnHCl revealed more stability of the circular form compared with the linear form.
Keywords
Intein; protein cyclization; green fluorescent protein; purification;
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1 Cormack, B. P., R. H. Valdivia, and S. Falkow. 1996. FACSoptimized mutants of the green fluorescent protein (GFP). Gene 173: 33-38.   DOI   ScienceOn
2 Evans, T. C. Jr., D. Martin, R. Kolly, D. Panne, L. Sun, I. Ghosh, et al. 2000. Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J. Biol. Chem. 275: 9091-9094.   DOI
3 Grantcharova, V. P., D. S. Riddle, and D. Baker. 2000. Longrange order in the src SH3 folding transition state. Proc. Natl. Acad. Sci. U.S.A. 97: 7084-7089.   DOI   ScienceOn
4 Iwai, H., A. Lingel, and A. Pluckthun. 2001. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276: 16548-16554.   DOI
5 Iwai, H. and A. Pluckthun. 1999. Circular beta-lactamase: Stability enhancement by cyclizing the backbone. FEBS Lett. 459: 166-172.   DOI   ScienceOn
6 Jeffries, C. M., S. C. Graham, P. H. Stokes, C. A. Collyer, J. M. Guss, and J. M. Matthews. 2006. Stabilization of a binary protein complex by intein-mediated cyclization. Protein Sci. 15: 2612-2618.   DOI   ScienceOn
7 Jermutus, L., M. Tessier, L. Pasamontes, A. P. van Loon, and M. Lehmann. 2001. Structure-based chimeric enzymes as an alternative to directed enzyme evolution: Phytase as a test case. J. Biotechnol. 85: 15-24.   DOI   ScienceOn
8 Otzen, D. E. and A. R. Fersht. 1998. Folding of circular and permuted chymotrypsin inhibitor 2: Retention of the folding nucleus. Biochemistry 37: 8139-8146.   DOI   ScienceOn
9 Thornton, J. M. and B. L. Sibanda. 1983. Amino and carboxyterminal regions in globular proteins. J. Mol. Biol. 167: 443-460.   DOI
10 Trabi, M. and D. J. Craik. 2002. Circular proteins - no end in sight. Trends Biochem. Sci. 27: 132-138.   DOI
11 Xu, M. Q. and T. C. Jr. Evans. 2001. Intein-mediated ligation and cyclization of expressed proteins. Methods 24: 257-277.   DOI   ScienceOn
12 Hofmann, A., H. Iwai, S. Hess, A. Pluckthun, and A. Wlodawer. 2002. Structure of cyclized green fluorescent protein. Acta Crystallogr. D Biol. Crystallogr. 58: 1400-1406.   DOI   ScienceOn
13 Wu, H., Z. Hu, and X. Q. Liu. 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.   DOI   ScienceOn
14 Andersen, J. B., C. Sternberg, L. K. Poulsen, S. P. Bjorn, M. Givskov, and S. Molin. 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64: 2240-2246.
15 Evans, T. C. Jr., J. Benner, and M. Q. Xu. 1999. The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins. J. Biol. Chem. 274: 18359-18363.   DOI
16 Grantcharova, V. P. and D. Baker. 2001. Circularization changes the folding transition state of the src SH3 domain. J. Mol. Biol. 306: 555-563.   DOI   ScienceOn
17 Rodriguez, E., Z. A. Wood, P. A. Karplus, and X. G. Lei. 2000. Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. Arch. Biochem. Biophys. 382: 105-112.   DOI   ScienceOn
18 Scott, C. P., E. Abel-Santos, M. Wall, D. C. Wahnon, and S. J. Benkovic. 1999. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. U.S.A. 96: 13638-13643.   DOI   ScienceOn
19 Hruby, V. J. 1982. Conformational restrictions of biologically active peptides via amino acid side chain groups. Life Sci. 31: 189-199.   DOI   ScienceOn
20 Williams, N. K., E. Liepinsh, S. J. Watt, P. Prosselkov, J. M. Matthews, P. Attard, J. L. Beck, N. E. Dixon, and G. Otting. 2005. Stabilization of native protein fold by intein-mediated covalent cyclization. J. Mol. Biol. 346: 1095-1108.   DOI   ScienceOn
21 Zhao, Z., W. Lu, B. Dun, D. Jin, S. Ping, W. Zhang, M. Chen, M. Q. Xu, and M. Lin. 2008. Purification of green fluorescent protein using a two-intein system. Appl. Microbiol. Biotechnol. 77: 1175-1180.   DOI   ScienceOn
22 Lehmann, M., C. Loch, A. Middendorf, D. Studer, S. F. Lassen, L. Pasamontes, A. P. van Loon, and M. Wyss. 2002. The consensus concept for thermostability engineering of proteins: Further proof of concept. Protein Eng. 15: 403-411.   DOI
23 Kanno, A., Y. Yamanaka, H. Hirano, Y. Umezawa, and T. Ozawa. 2007. Cyclic luciferase for real-time sensing of caspase 3 activities in living mammals. Angew. Chem. Int. Ed. Engl. 46: 7595-7599.   DOI   ScienceOn
24 Williams, N. K., P. Prosselkov, E. Liepinsh, I. Line, A. Sharipo, D. R. Littler, P. M. Curmi, G. Otting, and N. E. Dixon. 2002. In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. J. Biol. Chem. 277: 7790-7798.   DOI
25 Lehmann, M., L. Pasamontes, S. F. Lassen, and M. Wyss. 2000. The consensus concept for thermostability engineering of proteins. Biochim. Biophys. Acta 1543: 408-415.   DOI   ScienceOn
26 Lehmann, M., R. Lopez-Ulibarri, C. Loch, C. Viarouge, M. Wyss, and A. P. van Loon. 2000. Exchanging the active site between phytases for altering the functional properties of the enzyme. Protein Sci. 9: 1866-1872.   DOI   ScienceOn
27 Siebold, C. and B. Erni. 2002. Intein-mediated cyclization of a soluble and a membrane protein in vivo: Function and stability. Biophys. Chem. 96: 163-171.   DOI   ScienceOn