Kim Sanggil, Ko Wooseok, Park Hyunji, Lee Hyun Soo
Department of Chemistry, Sogang University.
Department of Chemistry, Sogang University;
J Vis Exp. 2016 Dec 23(118):54922. doi: 10.3791/54922.
There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).
目前有许多化学工具可用于将化学探针引入蛋白质中，以研究其结构和功能。一种有用的方法是通过基因导入含有生物正交官能团的非天然氨基酸来进行蛋白质偶联。本报告描述了一种用于位点特异性抗体偶联的详细方案。该方案包括含叠氮基氨基酸基因掺入的实验细节，以及通过应变促进的叠氮-炔环加成反应（SPAAC）进行的偶联反应。这种应变促进反应通过在生理pH值和温度下简单混合反应分子来进行，并且不需要额外的试剂，如铜（I）离子和铜螯合配体。因此，该方法对于一般蛋白质偶联和抗体药物偶联物（ADC）的开发将是有用的。
