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Directed Evolution of Thermostability 287temperature for 30 min. Remove supernatant, air-dry, and dissolve the pellet in 10to 20 µL of water.2. Transform desalted DNA into 100 µL of electrocompetent E. coli XL-1 Blue cellsusing 1.7 kV, 200 Ω, and 25 µF. Immediately after transformation, add 900 µL2YT and 1/100 volume of transformation salt stock.3. Transfer the cell suspensions in 10-mL glass test tubes and incubate for 60 to 70 minat 37°C with orbital shaking.4. Assess the transformation efficiency by plating a dilution series of each transformationmixture on LB/Cm agar.5. Use another aliquot of the transformed cells to estimate the level of selection pressurefor the following round by plating on LB/Cm agar plates supplemented with variousconcentrations of ampicillin (see Note 6). Take the highest ampicillin concentrationat which colonies can be observed as selection level for the subsequent round.6. For selection, plate the transformed cells on LB/Cm agar plates with the ampicillinconcentration determined in step 5 (see also Note 6). We used ampicillin concentrationsof 20 µg/mL, 100 µg/mL, and 200 µg/mL for the first, second, and thirdselection, respectively. The first selection round was performed in liquid medium,the other two rounds were selected on agar plates.The impaired truncation mutant N∆5-clone was revitalized by three roundsof directed evolution comprising random mutagenesis and DNA shuffling of aPCR product (as described in Subheading 3.5.). In three successive rounds,approx 19 × 10 3 , 147 × 10 3 , and 260 × 10 3 clones were generated and screenedfor survival on plates containing 20, 100, and 200 µg/mL ampicillin (see step 6),respectively, giving rise to approx 3, 600, and 7000 colonies.Table 1 summarizes all sequenced clones isolated in the course of directedevolution sorted by shuffling round (S1–S3). Additionally, Table 1 includes relativesolvent accessibilities and average side chain atomic temperature factors(taken from PDB 1btl) (37) for each of the substituted wt residues. The fiveclones sequenced after the second optimization round shared two mutations,M182T and T265M (numbering according to Ambler et al., ref. 38), whichwere already present after the first round.After the final optimization step, the library was pooled, and the maximumlevel of ampicillin resistance was determined on plates without IPTG, as describedin Subheading 3.4. (Fig. 3). Normal colony development of a significant numberof clones was seen up to 700 µg/mL, and, after prolonged incubation timesof 40 h, clones could even be detected on 1000 µg/mL. In contrast, the wt constructgrew only up to 500 µg/mL. Twenty-six clones were picked from the 800to 1000 µg/mL plates and sequenced. The 26 sequences corresponded to 15individual clones (Table 1). All clones shared two mutations, M182T andA224V. The M182T mutation has previously been demonstrated to compensatefor folding defects (39) and stability losses (29,40). The second mutation(A224V) prevailed at the increased selective pressure of the third round. This
Table 1Amino Acid Substitutions Selected in the Course of Directed Evolution of the N∆5 Deletion Mutant288Amino acid substitutions of the selected deletion mutants are shown with regard to wt sequence. Residues are numbered according to Ambler et al.(38). Mutants are grouped according to the round of directed evolution in which they were selected (S1–S3). The frequency of individual selected clonescontaining the same set of mutations is shown in brackets. The most prevalent amino acid substitutions of the third round mutants are shown in boldfacetype. % acc, relative solvent accessibility, the ratio between calculated and vacuum accessibility expressed as a percentage using the program What If (48);B-factors corresponding to the average of side-chain atoms were taken from the <strong>Protein</strong> Data Bank (PDB entry: 1btl).
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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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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
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: 286 Hecky et al.4. Analyze the resu
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.
Page 317 and 318: 304 Hecky et al.40. Wang, X., Minas
Page 319 and 320: 306 IndexCCalcium indicators, see C
Page 321 and 322: 308 Indexchemiocompetent cellprepar
Page 323 and 324: 310 Indexmaterials, 169, 170mutatio
Page 325: 312 IndexTerminal truncation, see a
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