Protein Engineering Protocols - Mycobacteriology research center

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115Fig. 1. Mapping group-specific residues on the structure of MutH. (Left) Part of the sequence alignment between the group of MutHproteins and the group of restriction endonucleases (REase). Residues conserved in both groups are shaded in gray with white lettering.Residues conserved only in the group of MutH proteins are shaded in gray with black lettering. (Right) In the structure of the E. coliMutH protein, group-specific residues are shown in gray. The positions of residues supposed to be involved in contacting the methylatedbases are indicated.
116 Friedhoff and PingoudFig. 2. Superposition of MutH with restriction endonucleases. (A) The two nucleotidesof the MunI recognition sequence corresponding to the two adenines of theMutH recognition sequence, d(GATC), are shown. The nucleotide with the asterisks(A*) corresponds to the adenine of the MutH recognition sequence in the lower strand,which can be methylated and then directs cleavage to the upper strand. (B,C)Quantitative analysis of the superposition of MutH with 11 different restriction endonucleases(BamHI, BglI, BglII, BsoBI, EcoRI, EcoRV, HincII, MunI, NaeI, NgoMIV,and PvuII). Shown are the average distances of the atoms of the MutH structure to theatom N6 (adenine), O6 (guanine), N4 (cytosine), or O4 (thymine) of the nucleotidecorresponding in position to the adenine in the upper strand (B) or lower strand (C),respectively, of the MutH recognition sequence. Note the minima, which indicate theproximity of residues, i.e., K48, F94, R184, and Y212, to the N6 of adenine or itsequivalent in both the upper and lower strand, respectively, in all of the superimposedDNA structures.3. GeneDoc (http://www.psc.edu/biomed/genedoc/).4. RasMol (http://www.openrasmol.org/).5. Swiss PDB Viewer (http://www.expasy.org/spdbv/).2.2. Site-Directed Mutagenesis of MutH1. pMQ402, a BAD18 derivative, is used as a bacterial expression plasmid (23).Under control of an arabinose promoter for inducible protein expression, it containsthe coding sequence for a His-tagged MutH protein, which can be purifiedby Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography. The plasmidharbors an ampicillin-resistance gene for selection.
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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 78 and 79: Design of Coiled Coil Structures 65
Page 84 and 85: 4Calcium Indicators Based on Calmod
Page 86 and 87: Protein-Based Ca 2+ Indicators 73Fi
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Page 94 and 95: Protein-Based Ca 2+ Indicators 8145
Page 96 and 97: 5Design and Synthesis of Artificial
Page 98 and 99: Design of Zinc Finger Proteins 853.
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Page 109 and 110: 96 Koide and Koidewhile retaining t
Page 111 and 112: 98 Koide and Koide5. M9-tryptone: M
Page 113 and 114: 100 Koide and Koidetarget-binding s
Page 115 and 116: 102 Koide and Koide4. Discard the s
Page 117 and 118: 104 Koide and Koideup to 1 mM for h
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
Page 126 and 127: Engineering Site-Specific Endonucle
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Page 140 and 141: 8Protein Library Design and Screeni
Page 142 and 143: Protein Library Design and Screenin
Page 148 and 149: 135Fig. 2. Excel worksheet describi
Page 150 and 151: 137Fig. 4. Excel worksheet describi
Page 168 and 169: 9Protein Design by Binary Patternin
Page 170 and 171: Protein Design by Binary Patterning
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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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