The methods used to generate diversity are described later in the chapter. in Visual Studio .NET

Printer Code 128 Code Set A in Visual Studio .NET The methods used to generate diversity are described later in the chapter.
The methods used to generate diversity are described later in the chapter.
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DIRECTED EVOLUTION OF MACROMOLECULAR BIOASSAY REAGENTS
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antibodies. Ribozymes (ribonucleotides with catalytic activity) are also targets for directed evolution; efforts are underway to obtain ribozymes with new catalytic activities. Examples of target molecules and their new/improved functions are presented in Table 8.1. 8.2. RATIONAL DESIGN AND DIRECTED EVOLUTION Rational protein design (or protein engineering) implies the use of knowledge to design or improve the characteristics of a biomolecule. Rational protein design is accomplished by methods such as site-directed mutagenesis and the insertion or deletion of DNA sequences to yield predictable changes in protein properties. Rational methods have been used successfully to improve enzymatic stability at high temperature and toward other denaturing physical or chemical agents. Rational methods are based on experimental evidence that the effect of single amino acid substitutions on protein stability can be well approximated as additive, distributed, and large independent interactions. Moreover, by comparison of homologous enzymes from thermophilic and nonthermophilic microorganisms, it is known that introduction of disul de bridges as well as increased numbers of proline residues increase protein stability. Rational design methods have been applied to a glucose dehydrogenase enzyme to improve thermal stability, EDTA tolerance, and substrate selectivity.1 Pyrroloquinoline quinone (PQQ) enzymes are found in Gram-negative bacteria. This family of enzymes uses PQQ as the prosthetic group. PQQ glucose dehydrogenase (PQQGDH) is a monomeric membrane-bound protein with a MW of 87 kDa. An improved version of this enzyme would have an important application in glucose sensors; the enzyme commonly used in these devices, GO, cannot be used under anaerobic conditions unless an oxidant is present to perform the function of O2. Yoshida et al.1 used homologous recombination of PQQGDH genes from Escherichia coli and Acinetobacter calcoaceticus, and previous knowledge to generate several chimera proteins. Their goals were to improve the PQQ binding constant (assayed as EDTA tolerance since a divalent ions is required for holoenzyme formation), to increase temperature stability, and to improve substrate selectivity. Their procedure is shown schematically in Figure 8.1. Seven genetic variants were produced from the original E. coli gene. Site-directed mutagenesis was used to substitute the His residue in position 775 to Asn (His775Asn). This mutation improves the substrate speci city but simultaneously causes a decrease in thermal stability. Some regions of A. calcoaceticus that are responsible for EDTA resistance and for thermal stability were inserted into the E. coli gene by homologous recombination. The chimeric genes obtained were used to transform E. coli cells, with each single clone expressing one variant of PQQ glucose dehydrogenase. The enzyme was isolated and assayed in a buffer containing PQQ to form the holoenzyme, and a colorimetric indicator. Enzymatic activity was assayed for different substrates, and the effect of EDTA and thermal stability were investigated. When
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RATIONAL DESIGN AND DIRECTED EVOLUTION
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A. calcoaceticus
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Bacterial cell PQQGDH gene Bacterial chromosome
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E. coli
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From bacterial cultures, cut and amplified PQQGDH genes
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Change single codon ( ) and homologous recombination (rational design, knowledge supported)
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Chimeric genes obtained Chimeric genes proteins expressed in transformed E. coli cells Partial purification of PQQGDH enzyme and assay
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EDTA tolerance
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Thermal stability
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Substrate specifity
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Figure 8.1. Steps followed for the construction (rational design) of an improved PQQGDH enzyme.
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compared with the original E. coli enzyme, the thermal stability of the best chimeras was eight times greater, and the selectivity toward glucose was improved. The EDTA tolerance was as high as the original enzyme from A. calcoaceticus. In contrast to rational design methods, where changes in protein properties may be predicted from sequence changes, random mutation methods have been introduced to empirically improve protein properties in the absence of a priori knowledge of structure function relationships. Error-prone polymerase chain reaction (PCR), for example, can be used to introduce approximately random mutations in ampli ed DNA sequences. The protein products of these mutations may then be screened to select successful or improved variants.2 Directed evolution may exploit both rational design and random mutation methods. Generally, a three-step cycle is reiterated until the desired properties are observed. In the rst step, genetic diversity is generated. The second step involves the physical or chemical linking of the genotype (nucleic acid) to the phenotype (the translated protein). In the nal step, the successful variants are identi ed and selected. An example of a typical directed evolution protocol is shown in Figure 8.2. The number of iterations of the three-step cycle required to achieve the desired changes in properties depends on two main factors. First, the so-called
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