The re assortment of mutations to produce favorable

The re-assortment of mutations to produce favorable combinations that can undergo natural selection is a critical component of biological evolution. This process can be simulated by directed evolution, which has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory uses error-prone DNA replication in vitro to generate genetic Fmoc-Ile-Wang resin and specific screens to identify protein variants with desired properties [10]. Where necessary, the genes for these variants can then be shuffled in a process akin to homologous recombination to achieve further improvements [10]. In several instances, chimeric enzymes with improved activity and stability have been isolated from libraries constructed using DNA shuffling [[11], [12], [13], [14]]. In other cases, the method resulted in libraries with either too many mutations in each gene [15] or too few crossovers [16] to be useful. DNA shuffling can take advantage of orthologous proteins to repurpose functional diversity from nature, i.e. in addition to using error-prone replication in vitro, it can be used to shuffle distantly related existing sequences to take advantage of the natural diversity that exists within a population and to provide a means to eliminate deleterious mutations that may accumulate in strains [17]. On the other hand, it is limited by the degree of sequence homology shared by the existing sequence variants [10].
Materials and methods
Results
Discussion Glucarpidase, the recombinant form of CPG2, has been used for more than two decades as a detoxifying agent for MTX and also in targeted cancer therapies such as ADEPT. However, its usefulness in both treatment regimens has been limited by its relatively low specific activity and the fact that patients often develop antibodies against it after repeated administration. In our previous study [29], we successfully produced two long-acting variants of glucarpidase, PEGylated glucarpidase and HSA-fused Glucarpidase. We demonstrated that both “biobetter” glucarpidases are less immunogenic and had prolonged half-lives relative to the native enzyme. However, the study did not address the question of the native enzymes relatively low specific activity. In the present work, we used mutagenesis techniques to produce further “biobetter” glucarpidase variants with improved activity. Following mutagenesis of the glucarpidase gene of Pseudomonas sp. strain RS-16 [30], approximately 73% of the clones retained enzyme activity, as indicated by the clear zones and yellow precipitate surrounding their colonies. However, there were very few that had ‘halos’ around colonies that were darker than that of the wild-type, which would be indicative either of more active glucarpidase variants or variants that over-produced the enzyme relative to the wild-type construct. DNA sequence analysis of the three mutants taken for further study indicated that each had a single point mutation leading to the alteration of a single amino acid at the protein level (Supplemental Fig. S3). The fact that only single point mutations were present suggests that the error-prone PCR may have contributed more than the DNA shuffling procedure to the production of these particular mutants. Alternatively, it is possible that combining two or more individual mutations into a single gene may have resulted in mutant enzymes with little or no enzyme activity.