COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.