个人信息Personal Information
副教授
博士生导师
硕士生导师
性别:男
毕业院校:大连理工大学
学位:博士
所在单位:生物工程学院
学科:生物化工
办公地点:生物工程学院547室
电子邮箱:renjun@dlut.edu.cn
Freezing-assisted synthesis of covalent C-C inked bivalent and bispecific nanobodies
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论文类型:期刊论文
发表时间:2019-01-14
发表刊物:ORGANIC & BIOMOLECULAR CHEMISTRY
收录刊物:SCIE、PubMed、Scopus
卷号:17
期号:2
页面范围:257-263
ISSN号:1477-0520
摘要:Bi-valent/specific antibodies are coming to the forefront of therapeutic and diagnostic applications for extending the functions of conventional antibodies. Nanobodies as building blocks, due to their small sizes, are prone to synthesizing these homo/hetero-dimers. However, the classical C-terminus to N-terminus (C-N) ligation manner for generating the dimer results in the inhibition of the antigen-binding capacity of the bivalent/specific antibodies. In this study, we designed and constructed several C-terminus to C-terminus (C-C) linked bivalent and bispecific nanobodies against the human beta 2-microglobulin via freezing, overcoming the biological function-disrupt raised by the C-N ligation. The nanobody modified by the formylglycine generating enzyme was ligated to a hydrazide or aminooxy bi-functionalized linker. During the process, we discovered that freezing significantly improved the efficiency of hydrazone or oxime formation between the linker and nanobodies, which could not take place at room temperature. By freezing from -10 to -20 degrees C, up to 50% yield of bivalent nanobodies was achieved within 24 h. The C-C linked nanobody-fusions maintained almost all of its binding activity and exhibited an increase by two orders of magnitudes in affinity kinetics, demonstrating the superiority of C-C over the C-N linking approach.