A biased mutation-assembling method-that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.
We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.
Loss of heterozygosity (LOH)is an important event of tumorigenesis. In gastric cancer, we found a novel region of LOH in chromosome 9q having about 800 kb deletions at 9q31.1. The microsatellite marker D9S938 in that region exhibiting the highest LOH frequency, 56.5%. In addition, the LOH at 9q31.1 did not show any relationship to either histologic types or stages of gastric cancers, and several genes were predicted in the remaining allele by in silico methods. These data suggest that the deletion at 9q31.1 would be common in both differentiated-type and undifferentiatedtype gastric cancers. Furthermore, this deletion was found in the primary tumors of early-stage gastric cancer, indicating that loss of function of predicted genes appears to be associated with the tumorigenesis of gastric cancer. © 2003 Wiley-Liss, Inc. Key words: gastric cancer; loss of heterozygosity; chromosome 9q; microsatellite markerLoss of heterozygosity (LOH) has occurred in many of the common cancers. 1 The LOH study is a comparison of polymorphilic loci in DNA and the finding of contiguous regions of tumor DNA where one allele is absent. These regions might be expected to contain suppressor genes. LOH analysis has identified large numbers of regions of chromosomal loss, but the number of suppressor genes that has been identified convincingly is small. The mechanism for the inactivation of a tumor suppressor gene that predisposes cancer is not only somatic mutations, but also promoter hypermethylation. 2 Gastric cancer is the most frequent malignancy of the gastrointestinal tract in Japanese and certain Southeast Asian populations and one of the leading causes of cancer mortality in the world. Chromosomal aberrations in gastric cancer have been extensively studied, and losses of many chromosomal loci have been discovered, including losses in 1p, 2p, 3p, 5q, 6q, 7q, 8q, 12q, 13p, 14q, 17p, 17q and 18q. 3-6 Among these, RUNX3 and BRCA1 were the examples that have recently been mapped onto chromosome 1p36.1 7 and 17q21, 8 respectively.We have detected a novel locus of LOH at chromosome 9q by a combination of in-gel competitive reassociation (IGCR) and array-based comprehensive genomic hybridization (array CGH) techniques in the diffuse-type gastric cancer (data not shown). LOH at 9q was previously reported in bladder cancer, 9 myeloma 10 and renal cell carcinoma, 11 but the gene in 9q whose loss might contribute to the development of cancer has not been identified yet. In our present study, we narrowed the LOH region in 9q by using microsatellite markers to identify a tumor suppressor gene involved in the gastric cancer development. Several candidate genes were mapped from RefSeq gene, mRNAs and expressed sequence tags (ESTs) and predicted by in silico analyses. MATERIAL AND METHODS Human genomic DNAsThirty-three human genomic DNA pairs from gastric cancer tissue and adjacent normal stomach tissue were obtained from the gastric cancer patients under informed consent in the Second
Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.
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