Directed Evolution of Thermostability 30113. Because the first seconds of the reaction are especially important for enzymes withvery short half-lifes, it is essential to start the measurement immediately afteradding the enzyme. In addition, instruments should be used that record at least onedata point per second.14. The enzyme decay was fitted using the three-parameter exponential decay fit∆A∆Atimplemented in SigmaPlot: = a + ⎡ e whereby a is a parameter∆t⎣ ⎢ ⎤ –λ ×∆t⎦⎥0accounting for basal and accumulated absorption of the nitrocefin solution andT 1/2= ln2/λ15. Depending on the number of data points per nanometer acquired, the parametersfor smoothing need to be adjusted. Alternatively, a running mean average can beused for smoothing. In addition to determining λ max, we also tested the shift of<strong>center</strong> of mass from 330 to 370 nm. Despite the use of more data points, thismethod did not necessarily give better results.16. The emission maximum (y obs) as a function of denaturant concentration, [D], wasdeconvoluted in the constituting signals of the three conformational states (y N,y I,y U) according to their fraction present (f N,f I,f U), which is described by y obs= y N×f N+ y I× f I+ y U× f U. The law of mass action, K NI= [I]/[N], K IU= [U]/[I], wascombined with mass conservation, [N] 0= [N] + [I] + [U], to calculate the fractions,f N= 1/(1 + K NI+ K NI× K IU), f I= K NI/(1 + K NI+ K NI× K IU), and f U= K NI×K IU/(1 + K NI+ K NI× K IU). Thermodynamic parameters were introduced using thelinear extrapolation method based on ∆G 0 = –RTlnK = ∆G 0 H 2 O– m × [D], assuminga linear dependence for all states (46).17. To account for small changes between the absolute maxima measured with the twofluorometers, the normalized fraction unfolded, f unfold= (y obs– y F)/(y U– y F), isgiven in the plot.References1. Jaenicke, R. and Böhm, G. (1998) The stability of proteins in extreme environments.Curr. Opin. Struct. Biol. 8, 738–748.2. Ladenstein, R. and Antranikian, G. (1998) <strong>Protein</strong>s from hyperthermophiles: stabilityand enzymatic catalysis close to the boiling point of water. Adv. Biochem. Eng.Biotechnol. 61, 37–85.3. Querol, E., Perez-Pons, J. A., and Mozo-Villarias, A. (1996) Analysis of proteinconformational characteristics related to thermostability. <strong>Protein</strong> Eng. 9, 265–271.4. Vieille, C. and Zeikus, G. J. (2001) Hyperthermophilic enzymes: sources, uses, andmolecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43.5. Pace, C. N., Grimsley, G. R., Thomson, J. A., and Barnett, B. J. (1988) Conformationalstability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds.J. Biol. Chem. 263, 11,820–11,825.6. Mason, J. M., Gibbs, N., Sessions, R. B., and Clarke, A. R. (2002) The influence ofintramolecular bridges on the dynamics of a protein folding reaction. Biochemistry41, 12,093–12,099.
302 Hecky et al.7. Thompson, M. J. and Eisenberg, D. (1999) Transproteomic evidence of a loop-deletionmechanism for enhancing protein thermostability. J. Mol. Biol. 290, 595–604.8. Vogt, G., Woell, S., and Argos, P. (1997) <strong>Protein</strong> thermal stability, hydrogen bonds,and ion pairs. J. Mol. Biol. 269, 631–643.9. Szilagyi, A. and Zavodszky, P. (2000) Structural differences between mesophilic,moderately thermophilic and extremely thermophilic protein subunits: results of acomprehensive survey. Structure Fold Des 8, 493–504.10. Karshikoff, A. and Ladenstein, R. (2001) Ion pairs and the thermotolerance of proteinsfrom hyperthermophiles: a “traffic rule” for hot roads. Trends Biochem. Sci.26, 550–556.11. Petsko, G. A. (2001) Structural basis of thermostability in hyperthermophilic proteins,or “there’s more than one way to skin a cat.” Methods Enzymol. 334, 469–478.12. Malakauskas, S. M. and Mayo, S. L. (1998) Design, structure and stability of ahyperthermophilic protein variant. Nat. Struct. Biol. 5, 470–475.13. Filikov, A. V., Hayes, R. J., Luo, P., et al. (2002) Computational stabilization ofhuman growth hormone. <strong>Protein</strong> Sci. 11, 1452–1461.14. Lehmann, M., Loch, C., Middendorf, A., et al. (2002) The consensus concept forthermostability engineering of proteins: further proof of concept. <strong>Protein</strong> Eng. 15,403–411.15. Stemmer, W. P. (1994) Rapid evolution of a protein in vitro by DNA shuffling.Nature 370, 389–391.16. Stemmer, W. P. (1994) DNA shuffling by random fragmentation and reassembly:in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91,10,747–10,751.17. Yang, F., Cheng, Y., Peng, J., Zhou, J., and Jing, G. (2001) Probing the conformationalstate of a truncated staphylococcal nuclease R using time of flight mass spectrometrywith limited proteolysis. Eur. J. Biochem. 268, 4227–4232.18. Van der Schueren, J., Robben, J., and Volckaert, G. (1998) Misfolding of chloramphenicolacetyltransferase due to carboxy-terminal truncation can be corrected bysecond-site mutations. <strong>Protein</strong> Eng. 11, 1211–1217.19. Sherwood, L. M. and Potts, J. T., Jr. (1965) Conformational studies of pancreaticribonuclease and its subtilisin-produced derivatives. J. Biol. Chem. 240, 3799–3805.20. Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., and Kanaya, S.(1994) A novel strategy for stabilization of Escherichia coli ribonuclease HI involvinga screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol.Chem. 269, 26,904–26,911.21. Trevino, R. J., Tsalkova, T., Kramer, G., Hardesty, B., Chirgwin, J. M., andHorowitz, P. M. (1998) Truncations at the NH2 terminus of rhodanese destabilizethe enzyme and decrease its heterologous expression. J. Biol. Chem. 273,27,841–27,847.22. Trevino, R. J., Gliubich, F., Berni, R., et al. (1999) NH2-terminal sequence truncationdecreases the stability of bovine rhodanese, minimally perturbs its crystalstructure, and enhances interaction with GroEL under native conditions. J. Biol.Chem. 274, 13,938–13,947.
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METHODS IN MOLECULAR BIOLOGY 352Pr
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M E T H O D S I N M O L E C U L A R
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PrefaceProtein engineering is a fas
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ContentsPreface ...................
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ContributorsKATJA M. ARNDT • Inst
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Contributors xiKAZUNARI TAIRA • D
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1Combinatorial Protein Design Strat
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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Combinatorial Protein Design Strate
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2Global Incorporation of Unnatural
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Incorporation of Unnatural Amino Ac
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Incorporation of Unnatural Amino Ac
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Incorporation of Unnatural Amino Ac
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Incorporation of Unnatural Amino Ac
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Incorporation of Unnatural Amino Ac
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3Considerations in the Design and O
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Design of Coiled Coil Structures 37
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Design of Coiled Coil Structures 39
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Design of Coiled Coil Structures 41
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Design of Coiled Coil Structures 43
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Design of Coiled Coil Structures 45
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Design of Coiled Coil Structures 57
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Design of Coiled Coil Structures 65
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Design of Coiled Coil Structures 67
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Design of Coiled Coil Structures 69
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4Calcium Indicators Based on Calmod
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Protein-Based Ca 2+ Indicators 73Fi
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Protein-Based Ca 2+ Indicators 7512
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Protein-Based Ca 2+ Indicators 77Fi
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Protein-Based Ca 2+ Indicators 79Fi
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Protein-Based Ca 2+ Indicators 8145
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5Design and Synthesis of Artificial
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Design of Zinc Finger Proteins 853.
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Design of Zinc Finger Proteins 873.
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Design of Zinc Finger Proteins 89pr
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Design of Zinc Finger Proteins 91Fi
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Design of Zinc Finger Proteins 932.
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96 Koide and Koidewhile retaining t
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98 Koide and Koide5. M9-tryptone: M
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100 Koide and Koidetarget-binding s
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102 Koide and Koide4. Discard the s
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104 Koide and Koideup to 1 mM for h
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106 Koide and Koideplate. Incubate
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108 Koide and Koide1. Perform steps
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7Engineering Site-Specific Endonucl
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Engineering Site-Specific Endonucle
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115Fig. 1. Mapping group-specific r
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8Protein Library Design and Screeni
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Protein Library Design and Screenin
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Protein Library Design and Screenin
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Protein Library Design and Screenin
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135Fig. 2. Excel worksheet describi
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137Fig. 4. Excel worksheet describi
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9Protein Design by Binary Patternin
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Protein Design by Binary Patterning
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Protein Design by Binary Patterning
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Protein Design by Binary Patterning
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10Versatile DNA Fragmentation and D
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NExT DNA Shuffling 169This chapter
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NExT DNA Shuffling 1713. Methods3.1
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NExT DNA Shuffling 17372°C, 4 min.
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NExT DNA Shuffling 175Fig. 2. Varia
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NExT DNA Shuffling 1773.5.1. Direct
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NExT DNA Shuffling 179Fig. 3. Reass
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181
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NExT DNA Shuffling 183likelihood of
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NExT DNA Shuffling 1853.9.2. Calibr
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NExT DNA Shuffling 187staining. Bec
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NExT DNA Shuffling 1894. Zhao, H.,
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11Degenerate Oligonucleotide Gene S
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Degenerate Oligonucleotide Gene Shu
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12M13 Bacteriophage Coat Proteins E
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Engineered M13 Bacteriophage Coat P
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222 Fujita et al.The methods that h
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224 Fujita et al.3. Streptavidin an
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226 Fujita et al.Fig. 2. (Continued
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228 Fujita et al.Fig. 3. (Continued
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230 Fujita et al.Fig. 3. (A) Schema
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232 Fujita et al.1. Add 2 µg mRNA
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234 Fujita et al.Innovative Bioscie
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236 Fujita et al.30. Kim, Y., Mlsa,
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238 Ghadessy and Holligeraffinity m
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240 Ghadessy and HolligerFig. 1. (A
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242 Ghadessy and HolligerFinally, t
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244 Ghadessy and Holliger2. Prepare
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246 Ghadessy and Holliger2. After 6
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248 Ghadessy and Holliger21. Oberho
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