Protein Engineering Protocols - Mycobacteriology research center

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Index 307parameters in creation and screening,biased libraries, 130, 131experimental biases, 133library representation, 132, 133size,design, 129, 130limits, 131, 132phage display, see Phage displayprobability calculations,library representation,equiprobable outcomes, 139–144, 149, 150experimental bias testing withχ 2 test, 148, 149, 152overview, 138, 139unequal probability outcomes,144–147, 151, 152software, 133Compartmentalized self-replication (CSR),principles, 239–242Taq polymerase engineering,bacterial expression, 243materials, 242, 243polymerase chain reaction,244, 246pull through, 245, 246quick screening of variants,245, 246setup, 243, 244, 246work-up of reaction,polyethylene glycol extraction,244–246quick work-up, 245Computational protein design,coiled coils, 59–61, 64combinatorial libraries, seeCombinatorial librariesdirected protein design, 4examples, 5, 6limitations, 6probabilistic protein design, 6, 7profile building, 8, 9sequence alignment, 8statistical theory of sequenceensembles, backboneflexibility, 9, 17, 18conformational entropy, 9, 10constrained optimization ofentropy, 10, 18energy functions, 10, 11reference energy, 12, 13rotamer and identity probabilities,13, 15solvation and hydrophobicenergy, 11, 12CSR, see Compartmentalizedself-replicationDDegenerate oligonucleotide geneshuffling (DOGS),chimeric gene amplification, 197, 199cloning of shuffled products, 199materials, 192, 193plate assays with Congo Red, 199,200, 202polymerase chain reaction,degenerate primers,design, 193–195, 197, 202features, 195–197, 201, 202gene-specific nested end primerdesign, 195overlap extension of genesegments, 197, 202principles, 192xylanase assay, 200, 201DHFR PCA, see Dihydrofolatereductase protein-fragmentcomplementation assayDihydrofolate reductase proteinfragmentcomplementationassay (DHFR PCA),principles, 251Ras-binding domain of Raf librarypreparation and selection,
308 Indexchemiocompetent cellpreparation, 266, 270clonal competition experiment,263, 264, 269clone isolation and sequencing,264, 269controls and stringency, 257,258, 261electroporation, 265, 266library cloning and recovery, 262,263, 268library synthesis, 261, 266, 267materials, 252–255overview, 251, 252protein purification andcharacterization, 264, 265,269, 270screening, 263, 268, 269steric requirements, 255, 257Directed evolution, seeCompartmentalized selfreplication;Nucleotide exchangeand excision technology; <strong>Protein</strong>thermostabilityDirected protein design, seeComputational protein designDNA polymerase,engineering,compartmentalized self-replication,see Compartmentalizedself-replicationexamples, 238rationale, 238repertoire selection, 238, 239functions, 237, 238DNA shuffling, see Degenerateoligonucleotide gene shuffling;Nucleotide exchange andexcision technology; <strong>Protein</strong>thermostabilityDOGS, see Degenerate oligonucleotidegene shufflingEElectroporation,monobody construction, 102M13 P8 engineering for phagedisplay, 214, 215, 218Ras-binding domain of Raf librarypreparation, 265, 266ELISA, see Enzyme-linkedimmunosorbent assayEndonucleases, see RestrictionendonucleasesEnzyme-linked immunosorbent assay(ELISA), phage displayanalysis, 216, 217FFibronectin type III domain, seeMonobodiesFluorescence resonance energy transfer(FRET),chameleons, see Chameleonsprinciples, 72FRET, see Fluorescence resonanceenergy transferGGene shuffling, see Degenerateoligonucleotide gene shufflingGFP, see Green fluorescent proteinGlutathione S-transferase (GST),purification after amino acid analogincorporation, 28, 32ribosome-inactivation displaysystem, see Ribosomeinactivationdisplay systemGreen fluorescent protein (GFP),calcium indicators, see Chameleonspurification after amino acid analogincorporation, 29, 32Growth hormone, phage display, 216, 217GST, see Glutathione S-transferase
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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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Page 289 and 290: 276 Hecky et al.improved half-life
Page 291 and 292: 278 Hecky et al.Fig. 1. (A) Scheme
Page 293 and 294: 280 Hecky et al.11. Reverse primer
Page 295 and 296: 282 Hecky et al.high variability in
Page 297 and 298: 284 Hecky et al.coding sequence for
Page 299 and 300: 286 Hecky et al.4. Analyze the resu
Page 301 and 302: Table 1Amino Acid Substitutions Sel
Page 303 and 304: 290 Hecky et al.Fig. 4. Structure o
Page 305 and 306: 292 Hecky et al.Fig. 5. Expression
Page 307 and 308: 294 Hecky et al.Table 2Kinetic Para
Page 309 and 310: 296 Hecky et al.The thermoactivity
Page 311 and 312: 298 Hecky et al.Fig. 8. Unfolding o
Page 313 and 314: 300 Hecky et al.for the first trans
Page 315 and 316: 302 Hecky et al.7. Thompson, M. J.
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Page 323 and 324: 310 Indexmaterials, 169, 170mutatio
Page 325: 312 IndexTerminal truncation, see a
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