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Ribosome-Inactivation Display System 229Fig. 3. (Continued)3.1.3. Construction of pStAv-mR and pGST-mR PlasmidsTo confirm that RTA helps to maintain a stable ribosome complex, in a controlexperiment, we made a gene for an inactive, mutant RTA by site-specificmutagenesis, changing functional amino acids as follows: glutamic acid 177to glutamine; arginine 180 to histidine; and glutamic acid 208 to aspartic acid.Mutation of these three amino acids in RTA completely abolished its activity
230 Fujita et al.Fig. 3. (A) Schematic drawing of the DNA constructs used for the model study ofRIDS. Streptavidin and GST genes were inserted between XbaI and NheI in the pLRS
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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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2Global Incorporation of Unnatural
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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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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 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 89pr
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Design of Zinc Finger Proteins 91Fi
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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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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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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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Page 196 and 197: NExT DNA Shuffling 183likelihood of
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Page 204 and 205: 11Degenerate Oligonucleotide Gene S
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Page 232: Engineered M13 Bacteriophage Coat P
Page 235 and 236: 222 Fujita et al.The methods that h
Page 237 and 238: 224 Fujita et al.3. Streptavidin an
Page 239 and 240: 226 Fujita et al.Fig. 2. (Continued
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Page 245 and 246: 232 Fujita et al.1. Add 2 µg mRNA
Page 247 and 248: 234 Fujita et al.Innovative Bioscie
Page 249 and 250: 236 Fujita et al.30. Kim, Y., Mlsa,
Page 251 and 252: 238 Ghadessy and Holligeraffinity m
Page 253 and 254: 240 Ghadessy and HolligerFig. 1. (A
Page 255 and 256: 242 Ghadessy and HolligerFinally, t
Page 257 and 258: 244 Ghadessy and Holliger2. Prepare
Page 259 and 260: 246 Ghadessy and Holliger2. After 6
Page 261 and 262: 248 Ghadessy and Holliger21. Oberho
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Page 267 and 268: 254 Campbell-Valois and Michnick7.
Page 269 and 270: 256 Campbell-Valois and MichnickFig
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Page 273 and 274: 260Fig. 3. BL21 pREP4 cells were co
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Page 289 and 290: 276 Hecky et al.improved half-life
Page 291 and 292: 278 Hecky et al.Fig. 1. (A) Scheme
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280 Hecky et al.11. Reverse primer
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282 Hecky et al.high variability in
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284 Hecky et al.coding sequence for
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286 Hecky et al.4. Analyze the resu
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Table 1Amino Acid Substitutions Sel
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290 Hecky et al.Fig. 4. Structure o
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292 Hecky et al.Fig. 5. Expression
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294 Hecky et al.Table 2Kinetic Para
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296 Hecky et al.The thermoactivity
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298 Hecky et al.Fig. 8. Unfolding o
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300 Hecky et al.for the first trans
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302 Hecky et al.7. Thompson, M. J.
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304 Hecky et al.40. Wang, X., Minas
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306 IndexCCalcium indicators, see C
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308 Indexchemiocompetent cellprepar
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310 Indexmaterials, 169, 170mutatio
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312 IndexTerminal truncation, see a
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