一步法固定化和纯化纤维素上的 CBD 融合内切酶 EndoS 用于抗体 Fc 糖基化修饰。
One-step immobilization and purification of genetic engineering CBD fusion EndoS on cellulose for antibodies Fc-glycan remodeling.
机构信息
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China.
出版信息
Bioorg Chem. 2019 Oct;91:103114. doi: 10.1016/j.bioorg.2019.103114. Epub 2019 Jul 12.
The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81-90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.
内切糖苷酶（EndoS 及其糖基合成酶突变体 D233A、D233Q）基因与纤维素结合域（CBD）融合，使用 pET-35b 载体进行表达，融合酶在大肠杆菌中成功表达。然后，开发了一种使用纤维素作为基质的一步法固定化和纯化 EndoS 酶的简化方法，在最佳条件下获得了 81-90%的高装载效率。纤维素固定化的 CBD-EndoS 和糖基合成酶突变体表现出高催化活性，并成功应用于两步法抗体 Fc N-糖基化改造，高效生成均一糖型的治疗性抗体。纤维素固定化的 CBD-EndoS 及其突变体（D233A 和 D233Q）在 4°C 下储存一个月时表现出优异的储存稳定性。可重复使用性研究表明，纤维素固定化的 CBD-EndoS 及其突变体可回收使用五次，而没有明显的活性损失。
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