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断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接

窦同璐, 陈浩, 曹津, 陈俊升, 朱建伟

窦同璐, 陈浩, 曹津, 陈俊升, 朱建伟. 断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接[J]. 中国药科大学学报, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515
引用本文: 窦同璐, 陈浩, 曹津, 陈俊升, 朱建伟. 断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接[J]. 中国药科大学学报, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515
DOU Tonglu, CHEN Hao, CAO Jin, CHEN Junsheng, ZHU Jianwei. Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area[J]. Journal of China Pharmaceutical University, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515
Citation: DOU Tonglu, CHEN Hao, CAO Jin, CHEN Junsheng, ZHU Jianwei. Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area[J]. Journal of China Pharmaceutical University, 2019, 50(5): 606-613. DOI: 10.11665/j.issn.1000-5048.20190515

断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接

基金项目: 国家自然科学基金资助项目(No.81773621)

Splicing reaction of split inteins between non-natural exteins containing amino acid sequences of antibody hinge area

  • 摘要: 内含肽是一种能够介导蛋白质分子从前体分子中自我切除,同时将其双侧的外显肽蛋白通过肽键连接起来的功能性蛋白质,其中断裂型内含肽在抗体连接领域有着广泛应用。但由于天然断裂型内含肽C端可特异性识别外显肽的前3位氨基酸序列——半胱氨酸、苯丙氨酸和天冬酰胺(CFN),因此在蛋白剪接反应后不可避免的会引入外源氨基酸。由于本课题组前期开发出的断裂内含肽介导的双特异性抗体药物装配平台也存在该缺陷,因此本研究尝试采用抗体铰链区的半胱氨酸、天冬氨酸和赖氨酸(CDK)取代“CFN”作为内含肽的识别位点,并构建突变型断裂内含肽Npu*GEP DnaE。结果表明,突变体可以识别“CDK”并成功发生了内含肽剪接反应。进而对影响该内含肽剪接反应平台的各因素如NaCl浓度、DTT浓度、pH和温度等进行了考察,结果表明在所考察的范围内断裂内含肽的剪接反应均可很好的进行。该内含肽剪接反应平台的建立将有助于减少外源氨基酸的引入,进一步拓展了内含肽底物宽泛性,为内含肽应用于抗体药物特别是双特异性抗体药物的装配提供了技术支持。
    Abstract: Intein is a functional protein that mediates self-cleavage from precursor protein and simultaneously connects the exteins on both sides of the intein via peptide bonds. Among all kinds of inteins, split intein has a wide range of applications in the field of antibody ligation. However, since the naturally split intein specifically recognizes the first three amino acid sequences “cysteine, phenylalanine, and asparagine”(CFN)of the extein, exogenous amino acids are inevitably introduced after the protein splicing reaction. In this study, the amino acid sequence “cysteine, aspartic acid, and lysine”(CDK)of the antibody hinge region was substituted for “CFN” as the recognition site for the intein and the split intein Npu DnaE was mutated into Npu*GEP DnaE. The results showed that the mutant could recognize “CDK” and the intein splicing reaction could successfully take place. The factors affecting the intein splicing reaction platform, such as pH, temperature and the concentration of NaCl and DTT were investigated in this study. The results showed that the splicing reaction of the mutant performed well, which indicated its potential usefulness in bispecific antibody assembly. In conclusion, the problem of introducing foreign amino acids was alleviated, the broadness of intein substrate was further expanded, and further technical support for the application of intein to the antibody assembly was provided.
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出版历程
  • 刊出日期:  2019-10-24

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