Protein Engineering Protocols - Mycobacteriology research center

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Design of Zinc Finger Proteins 873.1.4. CD MeasurementThe CD spectra of the zinc finger proteins are recorded on a Jasco J-720 spectropolarimeterin Tris-HCl buffer, pH 8.0, containing 50 mM NaCl in a capped 1-mm path-length cell at 20°C, under nitrogen. All spectra represent the average of 8to 16 scans. Spectra are baseline corrected and noise reduced using Jasco software.3.1.5. UV-VIS Absorption SpectroscopyUV-VIS absorption spectra are recorded on a Beckman Coulter DU7400 diodearray spectrophotometer at 20°C in 10 mM Tris-HCl buffer, pH 7.5, containing 50mM NaCl in a capped 1-cm path-length cell. Co(II)-substituted zinc finger complexesare obtained by titration with CoCl 2. The peptides are saturated with Co(II) inarbitrary conditions. All spectra are normalized by ε = A/(l •c), where ε is the extinctioncoefficient (per molar concentration per centimeter), l is the path-length of thecell (in centimeters), and c is the peptide concentration (in molar concentration).3.1.6. NMR ExperimentsIn the presence of 1.5 Eq of Zn(II) ion, the complex of single finger domainand Zn(II) is prepared at a concentration of 5 mM in 90% H 2O/10% D 2O andD 2O (25 mM Tris–d 11, pH 5.7). All of the NMR spectra are recorded on a JOELLambda-600 spectrometer.1. The nuclear Overhauser enhancement spectroscopy (NOESY) spectra areacquired with selective water presaturation (delays alternating with mutations fortailored excitation pulse) followed by the standard NOESY pulse train at 303 K,with mixing times of 100, 200, and 300 ms.2. The total correlation spectroscopy spectra are obtained with an 80-ms MLEV-17 spinlockduration, using water suppression by a gradient tailored excitation pulse at 303 K.Spectra are typically acquired with 24 scans per t1 valve, for 1024 t1 valves,and 2048 complex points are collected in the direct dimension. The free-inductiondecay in both dimensions is multiplied by the phase-shifted sine bell apodizationfunction, zero-filled, and Fourier transformed to yield 2048 by 2048 matrices.Sequential resonance assignments are determined by standard total correlationspectroscopy and NOESY procedures (8).3.2. Six- and Nine-Zinc Finger ProteinsThis section describes the design strategy and construction of multi-zinc fingersfor the recognition of long DNA sequences.3.2.1. Strategy of Protein DesignNovel six- and nine-zinc finger proteins, Sp1ZF6 and Sp1ZF9, were createdfrom the three-zinc finger motifs of the transcription factor, Sp1 (Fig. 2). These
88Fig. 2. Schematic representation of the artificial nine-zinc finger protein, Sp1ZF9. The DNA-binding site for the protein is shownabove of the nine-zinc finger illustration (see Note 2). The Krüppel-type linker sequences and other amino acids are represented bytheir one-letter codes.
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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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Page 84 and 85: 4Calcium Indicators Based on Calmod
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Page 96 and 97: 5Design and Synthesis of Artificial
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Page 109 and 110: 96 Koide and Koidewhile retaining t
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Page 121 and 122: 108 Koide and Koide1. Perform steps
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Page 140 and 141: 8Protein Library Design and Screeni
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137Fig. 4. Excel worksheet describi
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Protein Library Design and Screenin
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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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