Protein Engineering Protocols - Mycobacteriology research center

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PrefaceProtein engineering is a fascinating mixture of molecular biology, proteinstructure analysis, computation, and biochemistry, with the goal of developinguseful or valuable proteins. Protein Engineering Protocols will consider thetwo general, but not mutually exclusive, strategies for protein engineering. Thefirst is known as rational design, in which the scientist uses detailed knowledgeof the structure and function of the protein to make desired changes. The secondstrategy is known as directed evolution. In this case, random mutagenesisis applied to a protein, and selection or screening is used to pick out variantsthat have the desired qualities. By several rounds of mutation and selection,this method mimics natural evolution. An additional technique known as DNAshuffling mixes and matches pieces of successful variants to produce betterresults. This process mimics recombination that occurs naturally during sexualreproduction.The first section of Protein Engineering Protocols describes rational proteindesign strategies, including computational methods, the use of non-naturalamino acids to expand the biological alphabet, as well as impressive examplesfor the generation of proteins with novel characteristics. Although proceduresfor the introduction of mutations have become routine, predicting and understandingthe effects of these mutations can be very challenging and requiresprofound knowledge of the system as well as protein structures in general.Consequently, this section focuses on the question of how to design a proteinwith the desired properties, and examples are chosen to cover a wide range ofengineering techniques, such as protein–protein interactions, DNA binding,antibody mimics, and enzymatic activity.The second section of Protein Engineering Protocols deals with evolutionarytechniques. In contrast to rational design, directed evolution strategies donot require prior structural knowledge of a protein, nor is it necessary to beable to predict what effect a given mutation will have. Indeed, the results ofdirected evolution experiments are often surprising in that desired changes areoften caused by unexpected mutations. Several factors determine the successof such a strategy: the library design and quality, the choice of the method forevolution and/or DNA recombination, and the selection or screening method.Consequently, this second section of Protein Engineering Protocols providesinstructions to each of these steps, starting from general ideas of library designand statistical assessment of library quality. New methods for DNA shufflingv
vi Prefaceas well as different selection strategies are presented. Examples are given forthe evolution of different characteristics, such as protein folds, folding, thermostability,and enzyme activity.This volume provides a comprehensive guide to the methods used at everystage of the engineering process. It combines a thorough theoretical foundationwith detailed protocols and will be invaluable to all research workers in thearea, from graduate students to senior investigators. We would like to thank allauthors for their excellent contributions and Prof. John M. Walker for hiseditorial guidance, patience, and assistance throughout the editorial process.Katja M. ArndtKristian M. Müller
Page 1 and 2: METHODS IN MOLECULAR BIOLOGY 352Pr
Page 4 and 5: M E T H O D S I N M O L E C U L A R
Page 8 and 9: ContentsPreface ...................
Page 10 and 11: ContributorsKATJA M. ARNDT • Inst
Page 12: Contributors xiKAZUNARI TAIRA • D
Page 16 and 17: 1Combinatorial Protein Design Strat
Page 18 and 19: Combinatorial Protein Design Strate
Page 36 and 37: 2Global Incorporation of Unnatural
Page 38 and 39: Incorporation of Unnatural Amino Ac
Page 48 and 49: 3Considerations in the Design and O
Page 50 and 51: Design of Coiled Coil Structures 37
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Design of Coiled Coil Structures 43
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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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260Fig. 3. BL21 pREP4 cells were co
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262 Campbell-Valois and MichnickFig
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264 Campbell-Valois and Michnickvar
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276 Hecky et al.improved half-life
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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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