腺嘌呤碱基编辑器ABE8e多聚体的形成机制研究及抑制策略

Formation mechanism and suppression strategy of adenine base editor ABE8e aggregations

研究腺嘌呤碱基编辑器ABE8e(以下简称ABE8e)多聚化的成因,降低其多聚体的比例,为ABE8e的制备提供技术支持。首先,运用分子建模软件分析ABE8e的三维结构,了解多聚体的成因,在此基础上构建了相应的突变体ABE8e-T,并对其进行纯化,验证理论分析的正确性。在确认聚集原因后,为了降低ABE8e多聚体的比例,针对性地优化了ABE8e的缓冲体系。在结构信息的指导下,发现ABE8e二聚化界面中疏水区域的曝露导致了分子间聚集。突变体ABE8e-T(R64L/R74E)的纯化结果显示,二聚化界面进一步打开后,多聚体比例增多。缓冲体系优化结果表明,在现有缓冲体系中添加0.5 mol/L精氨酸,能中和分子表面的疏水性,从而降低了ABE8e的多聚体比例。结论:在ABE8e未形成二聚体的情况下,曝露了二聚化界面的疏水性区域,导致其分子间聚集,形成了多聚体。通过在缓冲液中添加精氨酸可以减弱分子间疏水作用,降低了多聚体的比例。因此,基于结构的分析方法可以精准地分析多聚体的成因,加速了下游工艺的开发,优化后的缓冲体系可为ABE8e的纯化和制备提供有用技术支持。

The cause of aggregations of adenine base editor ABE8e(hereinafter referred to as ABE8e)was studied,and the proportion of the aggregations was reduced,which provided technical support for the preparation of ABE8e.First of all,the three-dimensional structure of ABE8e was analyzed by molecular modeling software to find the cause of aggregations.On this basis,the corresponding mutant ABE8e-T was constructed and purified to verify the correctness of the theoretical analysis.After confirming the cause of aggregations,the buffer system of ABE8e was optimized in order to reduce the proportion of ABE8e aggregations.Under the guidance of structural information,it was found that ABE8e intermolecular aggregation mainly originated from the exposure of hydrophobic regions in the protein dimerization interface.The purification results of mutant ABE8e-T(R64L/R74E)showed an increase in the multimerization ratio after further opening of the dimerization interface.The optimization results of the buffer system showed that the addition of 0.5 mol/L arginine to the buffer system could neutralize the hydrophobicity of the molecular surface,thus reducing the proportion of aggregations.Conclusion:when ABE8e did not form a dimer,the hydrophobic region of the dimerization interface was exposed,leading to its intermolecular aggregations.By adding arginine to the buffer system,the intermolecular hydrophobic interaction was neutralized and the proportion of multimers reduced.Therefore,the structure-based analysis method could pinpoint the causes of aggregations and accelerate the development of downstream processes.The optimized buffer system could provide useful technical support for the purification and preparation of the base editor ABE8e.