Info

Wild-type GaOx

Asp

Gln

Tyr

Arg

Asn

His

Variant #1

Asp

Arg

Arg

Asn

Asn

His

Variant #2

Asp

Arg

Arg

Asp

Asn

His

Variant #3

Asp

Arg

Arg

Gln

Asn

His

Variant #4

Asp

Arg

Arg

His

Asn

His

Variant #5

Asp

Arg

Arg

Lys

Asn

His

Variant #6

Asp

Arg

Arg

Met

Asn

His

Variant #7

Asp

Arg

Arg

Ser

Asn

His

Variant #8

Asp

Arg

Asn

Lys

Asn

His

Variant #9

Asp

Arg

Glu

Lys

Asn

His

Variant #10

Asp

Arg

Glu

Ser

Asn

His

Variant #11

Asp

Arg

His

Asp

Asn

His

Variant #12

Asp

Arg

Lys

His

Asn

His

Variant #13

Asp

Arg

Lys

Tyr

Asn

His

Variant #14

Asp

Arg

Tyr

Asp

Asn

His

Variant #15

Asp

Arg

Tyr

Gln

Asn

His

Variant #16

Asp

Arg

Tyr

His

Asn

His

Variant #17

Asp

Arg

Tyr

Lys

Asn

His

Variant #18

Asp

Arg

Tyr

Met

Asn

His

Variant #19

Asp

Arg

Tyr

Ser

Asn

His

Variant #20

Asp

Asn

Arg

Asn

Asn

His

Variant #21

Asp

Asn

Arg

Asp

Asn

His

Variant #22

Asp

Asn

Arg

His

Asn

His

Variant #23

Asp

Asn

Arg

Lys

Asn

His

Variant #24

Asp

Asn

Lys

His

Asn

His

Variant #25

Asp

Asn

Tyr

His

Asn

His

Variant #26

Asp

Asp

Arg

Asn

Asn

His

Variant #27

Asp

Gln

Arg

Gln

Asn

His

Variant #28

Asp

Gln

Arg

Lys

Asn

His

Variant #29

Asp

Gln

Arg

Ser

Arg

His

Variant #30

Asp

Gln

Asn

Gln

Asn

His

Variant #31

Asp

Gln

Lys

Gln

Asn

His

Variant #32

Asp

Gln

Lys

Lys

Asn

His

Variant #33

Asp

Gln

Tyr

Arg

Asn

His

Variant #34

Asp

Gln

Tyr

Lys

Asn

His

Variant #35

Asp

Glu

Arg

Gln

Asn

His

Variant #36

Asp

Glu

Arg

Lys

Asn

His

Variant #37

Asp

Glu

Arg

Ser

Asn

His

Variant 38

Asp

Glu

Lys

Lys

Asn

His

TABLE 4.1A (continued)

Top 48 Predicted Sequences Spanning Residues 324-334 in Galactose Oxidase

TABLE 4.1A (continued)

Top 48 Predicted Sequences Spanning Residues 324-334 in Galactose Oxidase

Variant #39

Asp

Glu

Tyr

Gln

Asn

His

Variant #40

Asp

Lys

Arg

Gln

Asn

His

Variant #41

Asp

Lys

Arg

Glu

Asn

His

Variant #42

Asp

Lys

Lys

Glu

Asn

His

Variant #43

Asp

Lys

Tyr

Gln

Asn

His

Variant #44

Asp

Lys

Tyr

Glu

Asn

His

Variant #45

Asp

Ser

Arg

Lys

Asn

His

Variant #46

Gly

Arg

Tyr

Ile

Asn

His

Variant #47

Ser

Arg

Arg

Ser

Asn

His

Variant #48

Ser

Gln

Arg

Asp

Arg

Glu

Note: Program Rosetta (Kuhlman and Baker 2000; Meiler and Baker 2006) was used to predict combinations of amino-acid substitutions near the active site of galactose oxidase (Figure 4.6) to change enzyme specificity from galactose to glucose. This table lists the substitutions between D324 and H334 in the 48 variants predicted to be the most consistent with glucose binding (S. Lippow, unplub-lished results). Table 4.1B compares different library-construction methods that can be used to construct libraries that contain these 48 variants and the minimal number of library clones required to sample those 48 variants.

Note: Program Rosetta (Kuhlman and Baker 2000; Meiler and Baker 2006) was used to predict combinations of amino-acid substitutions near the active site of galactose oxidase (Figure 4.6) to change enzyme specificity from galactose to glucose. This table lists the substitutions between D324 and H334 in the 48 variants predicted to be the most consistent with glucose binding (S. Lippow, unplub-lished results). Table 4.1B compares different library-construction methods that can be used to construct libraries that contain these 48 variants and the minimal number of library clones required to sample those 48 variants.

are synthesized in the same physical oligonucleotide. The complex mixture of self-annealed hairpins that results is then exposed to MutS, and hairpins containing mismatches are removed as in the case of single oligonucleotide pairs (Figure 4.4).

Scanning Mutagenesis

The scanning-mutagenesis family of methods generates libraries that exhaustively sample a very simple library design: a collection of single point mutations at every position in the protein, or at least at every position in the region of interest. The earliest form of this method, alanine scanning (Cunningham and Wells 1989; Clackson and Wells 1995; Weiss et al. 2000), mutates each residue in turn to alanine, thus interrogating mostly the effect of a loss of chemical functionality at different positions in the sequence. In order to capture the information inherent in the loss-of-function phenotype of mutants in an alanine-scanning library, a high-throughput screen that obtains information on each individual mutant is required. The results from screening an alanine-scanning library are typically used to focus the design of a more thorough library synthesized by in-depth diversification (see the section titled "Site-Directed Diversification" in this chapter), focusing on the residues found to be sensitive to alanine scanning.

More complex scanning libraries can be generated by mutating each codon position of interest to a number of different codons. Typically, degenerate synthetic oligonucle-otides NNN, NNS, or NNK (defined in the preceding section) are used to replace each

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