Protein Engineering Protocols - Mycobacteriology research center

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Index 309HHelix, see Binary patterning; CoiledcoilMMass spectrometry (MS), amino acidanalog-incorporated proteinanalysis with liquidchromatography/massspectrometry, 30Monobodies,applications, 95biotinylation, 107, 108fibronectin type III domain scaffold,95, 96preparation,cloning, 107combinatorial libraryconstruction,double-stranded DNAsynthesis, 101, 108electrocompetent cellpreparation, 101, 102, 108electroporation for phagemidDNA, 102mutagenic oligonucleotidepreparation, 101, 108uracil single-stranded phagepreparation, 100, 101yeast library construction,102, 103expression, 107, 108materials, 96, 98, 99, 108phage display library sorting,panning, 103, 104phage clone characterization, 104purification, 107yeast two-hybrid system,bait plasmid preparation,104, 105liquid β-galactosidase assay, 106screening, 105, 106specificity test of isolatedmonobodies, 106MS, see Mass spectrometryMutH,DNA mismatch repair, 112, 113DNA nicking and cleavage assays,114, 119, 121features, 113materials for mutant preparation andcharacterization, 114, 116, 117methylation status sensing atd(GATC) sites, 113, 114purification of mutant proteins,119, 121sequence alignment studies insubstrate recognition,base-contacting residueidentification, 118DNA-binding residueidentification, 118, 121overview, 113programs, 114, 116site-directed mutagenesis, 119, 121NNExT, see Nucleotide exchange andexcision technologyNMR, see Nuclear magnetic resonanceNuclear magnetic resonance (NMR),zinc finger proteins, 87Nucleotide exchange and excisiontechnology (NExT),advantages, 169cloning, 171crossover rate analysis, 180, 182directed evolution, 167enzymatic digestion and chemicalcleavage, 173–175, 187gene fragments,denaturing polyacrylamide ureagel electrophoresis, 175, 176,187, 188mean fragment length analysis,180, 182purification,preparative gels, 177, 178silica-based resin afterpiperidine cleavage, 177, 188quantification, 178, 188reassembly and amplification,178–180, 188
310 Indexmaterials, 169, 170mutation rate analysis, 180, 182NExTProg for fragmentationprediction,calibration and comparison withexperimental results, 185–187mathematics, 183, 188overview, 182, 183principles, 168uridine-exchange polymerase chainreaction, 171, 173, 187PPCA, see Protein-fragmentcomplementation assayPCR, see Polymerase chain reactionPeptide Velcro hypothesis, see Coiled coilPhage display,coiled coil selection, 59library screening, 250M13 coat proteins,P8 engineering for improveddisplay,dU-single-stranded DNAtemplate preparation, 210,211, 217electroporation and phagepropagation, 214, 215, 218heteroduplex closed circularDNA synthesis, 212–214, 218library design, 209, 210materials, 206–208overview, 206, 208selection and analysis ofmutants, 216, 217stop template phagemid, 210, 217types for fusion, 205, 208monobody library sorting,panning, 103, 104phage clone characterization, 104ribosome-inactivation displaysystem, see Ribosomeinactivationdisplay systemPolymerase chain reaction (PCR),compartmentalized self-replication,244, 246nucleotide exchange and excisiontechnology and uridineexchangepolymerase chainreaction, 171, 173, 187random mutagenesis, 285, 286, 300ribosome-inactivation displaysystem, 233Protein-fragment complementationassay (PCA),coiled coil selection, 58, 59dihydrofolate reductase, seeDihydrofolate reductaseprotein-fragmentcomplementation assayprinciples, 251steric requirements, 255, 257Protein thermostability,compensatory mutation by directedevolution, 277rational design, 276rationale for engineering, 275, 276terminal truncation–directedevolution–re-elongation forenzyme enhancement,DNA shuffling and randommutagenesis, 285, 286, 300enzyme assay,kinetic parameterdetermination, 294spectrophotometric assay,293, 301thermoactivity profiling, 294,296, 301full-length mutant construction,290, 291β-lactamase as model system,281, 282materials, 279–281plasmid design, 282–284principles, 275–277, 279
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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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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
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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
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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: 308 Indexchemiocompetent cellprepar
Page 325: 312 IndexTerminal truncation, see a
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