Protein Engineering Protocols - Mycobacteriology research center

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Index 311protein purification,anion-exchangechromatography, 293, 300cell lysis, 292expression, 291, 292, 300hydrophobic interactionchromatography, 293, 300immobilized metal affinitychromatography, 292, 293phenylboronate affinitychromatography, 292terminal truncation,assay of effects, 284, 300design, 282transformation and in vivoselection of mutant libraries,286, 287, 289, 290, 300urea-induced unfolding studies,296–301RRaf, see Ras-binding domainRas-binding domain (RBD), Raf librarypreparation and selection,chemiocompetent cell preparation,266, 270controls and stringency, 257, 258, 261dihydrofolate reductase proteinfragmentcomplementation assay,clonal competition experiment,263, 264, 269clone isolation and sequencing,264, 269principles, 251protein purification andcharacterization, 264, 265,269, 270screening, 263, 268, 269electroporation, 265, 266library cloning and recovery, 262,263, 268library synthesis, 261, 266, 267materials, 252–255overview, 251, 252steric requirements, 255, 257RBD, see Ras-binding domainRestriction endonucleases,applications, 111MutH engineering, see MutHspecificity, 111, 112substrate recognition, 112RIDS, see Ribosome-inactivationdisplay systemRibosome-inactivation display system(RIDS),advantages over cell-dependentdisplay systems, 221, 222expression plasmid construction,glutathione S-transferase expressionplasmid, 228, 229, 231streptavidin expression plasmid,228, 229, 231universal plasmid, 224, 225materials, 223, 224principles, 222, 223selection,affinity selection of ribosome–RNA–protein complex, 231–233messenger RNA isolation, 231–233polymerase chain reaction, 233reverse transcription, 231, 232SStreptavidin, see Ribosome-inactivationdisplay systemTTaq polymerase, compartmentalizedself-replication forengineering,bacterial expression, 243materials, 242, 243polymerase chain reaction, 244, 246principles, 239–242pull through, 245, 246quick screening of variants, 245, 246setup, 243, 244, 246work-up of reaction,polyethylene glycol extraction,244–246quick work-up, 245
312 IndexTerminal truncation, see also <strong>Protein</strong>thermostability,compensatory mutation by directedevolution, 277protein tolerance, 276, 277Thermostability, see <strong>Protein</strong>thermostabilityUUnnatural amino acids, see Amino acidanalogsYYeast two-hybrid system,coiled coil selection, 59monobody screening,bait plasmid preparation, 104, 105liquid β-galactosidase assay, 106screening, 105, 106specificity test of isolatedmonobodies, 106ZZinc finger proteins,characterization,circular dichroism, 87absorption spectroscopy, 87nuclear magnetic resonance, 87design and synthesis,AT-recognizing zinc fingerproteins, 91, 92DNA-bending zinc fingerproteins, 89, 92Escherichia coli growth andinduction, 85, 86expression vector preparation, 85(His)4-type zinc finger proteins,89–91materials, 84purification, 86rationale, 83six- and nine-zinc finger proteins,87, 89general features, 83, 84
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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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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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250 Campbell-Valois and Michnickscr
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252 Campbell-Valois and Michnickfol
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254 Campbell-Valois and Michnick7.
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256 Campbell-Valois and MichnickFig
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258 Campbell-Valois and Michnickres
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Page 289 and 290: 276 Hecky et al.improved half-life
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Page 293 and 294: 280 Hecky et al.11. Reverse primer
Page 295 and 296: 282 Hecky et al.high variability in
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Page 301 and 302: Table 1Amino Acid Substitutions Sel
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Page 307 and 308: 294 Hecky et al.Table 2Kinetic Para
Page 309 and 310: 296 Hecky et al.The thermoactivity
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Page 315 and 316: 302 Hecky et al.7. Thompson, M. J.
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Page 319 and 320: 306 IndexCCalcium indicators, see C
Page 321 and 322: 308 Indexchemiocompetent cellprepar
Page 323: 310 Indexmaterials, 169, 170mutatio
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