Protein Engineering Protocols - Mycobacteriology research center

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Monobodies 97Fig. 1. (A) A schematic drawing of the FNfn10 structure. The locations of β-strandsA to G and loops connecting them are shown. (B) The amino acid sequence of FNfn10shown in its β-strand topology. Residues in the loops have gray background. Hydrogenbonds found in the structure are shown with arrows and lines. (C) The nucleic acidsequence of a synthetic gene for FNfn10 that we have used in our studies.
98 Koide and Koide5. M9-tryptone: Mix 6.4 g of Na 2H PO 4•7H 2O, 1.5 g of KH 2PO 4, 0.25 g of NaCl, 0.5 gof NH 4Cl, and 5 g of bacto-tryptone in 500 mL water, and autoclave. Cool to 50°Cand add 5 mL of 50% glucose, 1 mL of 1 M MgSO 4, and 50 µL of 1 M CaCl 2.6. Final concentrations of antibiotics are: 100 µg/mL ampicillin (Ap); 30 µg/mLchloramphenicol; and 10 µg/mL tetracycline (Tc).7. Minimal kanamycin sulfate (Km) plates: Mix 6 g bacto-agar in 320 mL of waterand autoclave, and then cool to 50°C. In a separate bottle, add 4.2 g K 2HPO 4, 1.8 gKH 2PO 4, 0.4 g (NH 4) 2SO 4, 0.2 g sodium citrate, and 64 mL of water, and autoclave,then cool to 50°C. Add 64 mL of the media to the bottle, 4 mL of 10 mg/mLuracil, 4 mL of 10 mg/mL histidine, 4 mL of 10 mg/mL leucine, 1.6 mL of 50%glucose, 0.32 mL of 1 M MgSO 4, 0.66 mL of 30 mg/mL Km, and 20 µL of thiamine100 mg/mL.8. Yeast extract-peptone-dextrose (YPD): 20 g bacto-peptone, 10 g yeast extract, and20 g glucose in 1 L water. For plates, add 20 g/L bacto-agar.9. Yeast complete (YC) glucose leu– his– trp– ura–: For 1 L, mix 1.2 g of YeastNitrogen Base w/o amino acids w/o ammonium sulfate (Difco), 5 g (NH 4) 2SO 4,10 g succinic acid, 6 g NaOH, 0.6 g complete supplement mixture-leu-his-trp-ura(Q-BIOgene), 0.1 g of cysteine, and 960 mL of water and autoclave. Add 40 mLof sterile 50% glucose and mix well. For plates, add 20 g/L bacto-agar.10. YC Glc his– ura– trp–: add 0.1 g of leucine to YC Glc leu– his– trp– ura– mediabefore autoclave.11. YC Glc trp–: add 0.1 g of leucine, 0.05 g of histidine, and 0.1 g of uracil to YCGlc leu– his– trp– ura– media before autoclaving.12. YC Glc his– ura–: add 0.1 g of leucine to YC Glc leu– his– trp– ura– media beforeautoclaving and use 910 mL of water instead of 960 mL. Add 50 mL of sterile2mg/mL tryptophan solution after autoclave.13. YC galactose raffinose leu– his– trp– ura–: replace glucose of YC Glc leu– his–trp– ura– media with 100 mL of sterile 20% galactose and 50 mL of sterile 20%raffinose. Use 850 mL of water instead of 960 mL.14. YC Gal Raf his– trp– ura–: add 0.1 g of leucine to YC Gal Raf leu– his– trp– ura–media before autoclaving.2.2. Reagents1. Polyethylene glycol (PEG)/NaCl solution: Mix 66.6 g of PEG8000, 78 g of NaCl,and 317 mL of H 2O, and autoclave.2. HEPES buffer: 1 mM HEPES, pH 7.0.3. Coating solution: 20 µg/mL ligand in 0.1 M NaHCO 3, pH 8.6.4. Tris-buffered saline (TBS): 50 mM Tris-HCl and 150 mM NaCl, pH 7.5.5. TBS–Tween-20 (TBST): TBS and 0.5% (v/v) Tween-20.6. 5% sterile bovine serum albumin (BSA): filter sterilize 5% BSA in water and storeat 4°C.7. TBST–BSA: TBST with 1 mg/mL BSA.8. Acid elution buffer: 0.1 N HCl adjusted to pH 2.2 with glycine, and 1 mg/mL BSA.9. 2 M Tris-base.
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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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Page 60 and 61: Design of Coiled Coil Structures 47
Page 84 and 85: 4Calcium Indicators Based on Calmod
Page 86 and 87: Protein-Based Ca 2+ Indicators 73Fi
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Page 96 and 97: 5Design and Synthesis of Artificial
Page 98 and 99: Design of Zinc Finger Proteins 853.
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Page 119 and 120: 106 Koide and Koideplate. Incubate
Page 121 and 122: 108 Koide and Koide1. Perform steps
Page 124 and 125: 7Engineering Site-Specific Endonucl
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Page 128 and 129: 115Fig. 1. Mapping group-specific r
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Page 140 and 141: 8Protein Library Design and Screeni
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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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