COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

COBRE P3：人性化海藻酸盐解聚酶

• 批准号: 7960371
• 负责人: Karl E Griswold
• 金额: $ 26.74万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960371
• 关键词: Alginates; Biological Response Modifier Therapy; Biopolymers; Carbohydrates; Centers of Research Excellence; 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; Lung diseases; Molecular; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 囊性纤维化的过早死亡通常是由于患者气道的慢性铜绿假单胞菌感染。与囊性纤维化相关的铜绿假单胞菌的粘液样菌株的特征在于它们产生外泌多糖藻酸盐，这是一种已知的致病因子。藻酸盐裂解酶是降解藻酸盐聚合物的酶，并且已经显示出用于囊性纤维化的对症治疗的巨大前景。 然而，这些生物催化剂的微生物来源使它们倾向于高水平的免疫原性，并使它们在临床环境中的实用性受到质疑。 不幸的是，与许多微生物生物酶候选物一样，藻酸盐裂解酶没有确切的人类对应物。 然而，人类基因组编码许多具有类似催化功能的酶，即基于碳水化合物的生物聚合物的降解。 显示出相似催化功能但不同底物选择性的人酶的存在表明藻酸盐裂解酶治疗活性可以通过工程改造人碳水化合物水解酶以作用于藻酸盐底物来去免疫化。 具体而言，具有改变的底物选择性的人酶的定向进化可以产生有效降解藻酸盐治疗靶标的生物催化剂，但由于其人源性，不太可能在人类患者中引起有害的免疫应答。 将采用定向进化来改变人溶菌酶的底物选择性以产生人源化藻酸盐解聚酶。 将这些酶的活性与真实的细菌藻酸盐裂解酶进行比较，并相对于野生型人溶菌酶模板评估工程化酶的免疫原性。