COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

COBRE P3：人性化海藻酸盐解聚酶

• 批准号: 8167472
• 负责人: Karl E Griswold
• 金额: $ 26.53万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167472
• 关键词: Alginates; Biological Response Modifier Therapy; Biopolymers; Carbohydrates; Chronic; Clinical; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Engineering; Enzymes; Exhibits; Funding; Grant; Human; Human Genome; Hydrolase; Immune response; Infection; Institution; Muramidase; Patients; Polymers; Premature Mortality; Production; Pseudomonas aeruginosa; Relative (related person); Research; Research Personnel; Resources; Source; Therapeutic; United States National Institutes of Health; base; directed evolution; enzyme activity; immunogenicity; microbial; mucoid; poly(beta-D-mannuronate) lyase; therapeutic target

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. The mucoid strains of P. aeruginosa associated with cystic fibrosis are characterized by their production of the exopolysaccharide alginate, a known pathogenic factor. Alginate lyases are enzymes that degrade the alginate polymer, and have shown great promise for symptomatic treatment of cystic fibrosis. However, the microbial origin of these biocatalysts predisposes them towards high level immunogenicity, and brings into question their utility in a clinical setting. Unfortunately, as is the case with numerous microbial biotherapeutic candidates, alginate lyases have no exact human counterpart. However, the human genome encodes numerous enzymes with similar catalytic functions, i.e. degradation of carbohydrate-based biopolymers. The existence of human enzymes exhibiting similar catalytic functions but different substrate selectivities suggests that the alginate lyase therapeutic activity could be deimmunized by engineering a human carbohydrate hydrolase to act on the alginate substrate. Specifically, directed evolution of a human enzyme with altered substrate selectivity could yield a biocatalyst that efficiently degrades the alginate therapeutic target, but because of its human origin would be less likely to illicit a deleterious immune response in human patients. Directed evolution will be employed to alter the substrate selectivity of human lysozyme to generate a humanized alginate depolymerase. The activity of these enzymes will be compared to an authentic bacterial alginate lyase, and the immunogenicity of the engineered enzymes will be assessed relative to the wild type human lysozyme template.

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