断裂型内含肽Npu DnaE的C端序列赖氨酸突变对其剪接活性的影响  

Effect of Lysine Mutation in the C-Terminal Sequence of Npu DnaE Split Intein on the Splicing Activity

作　　者：张敬仪 朱建伟 张宝红 ZHANG Jingyi;ZHU Jianwei;ZHANG Baohong(Engineering Research Center of Cell&Therapeutic Antibody,MOE,School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240)

机构地区：[1]上海交通大学药学院,细胞工程及抗体药物教育部工程研究中心,上海200240

出　　处：《中国医药工业杂志》2022年第12期1719-1725,共7页Chinese Journal of Pharmaceuticals

摘　　要：断裂型内含肽Nostoc punctiforme(Npu)DnaE作为一种为构建抗体-药物偶联物(ADC)过程中实现定点偶联的工具,其C端序列中的赖氨酸对其剪接活性有较大影响。该研究对断裂型内含肽Npu DnaE C端序列中的赖氨酸进行定点突变,得到了5种氨基酸(精氨酸、谷氨酰胺、甘氨酸、谷氨酸和甲硫氨酸)取代的多肽序列,以探究不同氨基酸取代对其剪接活性的影响。结果显示分别由精氨酸、谷氨酰胺和甲硫氨酸3种氨基酸取代赖氨酸的内含肽C端序列保持了剪接活性,其中精氨酸取代的内含肽C端序列剪接效率较高。采用精氨酸取代的内含肽C端序列制备了ADC HER2-Lc-SMCCDM1,经检测其保有抗原亲和力和抗肿瘤活性。该研究结果为ADC的定点偶联和基于内含肽设计、改造蛋白质或多肽连接合成相关研究提供了技术支撑。The split intein,Nostoc punctiforme(Npu)DnaE,is a site-specific conjugating tool for antibody-drug conjugates(ADC).Its splicing activity is affected by the lysine residues in the C-terminal sequence.This study focused on the splicing activities of different lysine mutants in the C-terminal sequence of Npu DnaE.Five amino acid(arginine,glutamine,glycine,glutamic acid and methionine)mutants were performed to explore the effects of different site-directed mutagenesis on lysine in the C-terminal sequence of the split intein Npu DnaE.The results showed that substitutions of lysine in the C-terminus by arginine,glutamine and methionine maintained splicing activity,and the arginine-substituted C-terminus exhibited the higher splicing efficiency.The ADC,HER2-Lc-SMCC-DM1,was further produced by split intein with arginine-substituted C-terminus,and both the antigen affinity and bioactivity were retained.This research provided technical support for site-specific ADC and protein/peptide synthesis based on the application of the intein.

关 键 词：Npu DnaE 断裂型内含肽 赖氨酸突变 剪接活性 抗体-药物偶联物 

分 类 号：Q816[生物学—生物工程]