山黧豆β-腈基丙氨酸合成酶的原核表达及蛋白纯化
投稿时间:2021-06-19  修订日期:2021-08-20  点此下载全文
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作者单位邮编
贾海燕 陇东学院生命科学与技术学院 745000
李辰浩 西北农林科技大学生命科学学院 712100
宋瑶瑶 西北农林科技大学生命科学学院 712100
刘凤娟 西北农林科技大学生命科学学院 712100
焦成瑾 天水师范学院生物工程与技术学院 741000
徐全乐 西北农林科技大学生命科学学院 712100
基金项目:陕西省自然科学基金(2020JM-167),大学生创新创业训练计划项目(S202010712196),中央高校基本科研业务费专项资金重点研究基地建设项目(lzujbky-2021-kb05)
中文摘要:β-N-草酰-L-α,β-二氨基丙酸(β-N-oxalyl-L-α,β-diaminopropionic acid,β-ODAP)是山黧豆(Lathyrus sativus)中的神经激活性物质,在低浓度时具有神经保护作用,高浓度时具有神经毒性,是山黧豆中毒的主要因素。代谢组学和转录组学的研究表明,β-ODAP的生物合成与硫代谢等基础代谢途径密切相关。其中,β-腈基丙氨酸合成酶(β-cyanoalanine synthase,β-CAS)可以催化异噁唑-5-酮与Cys反应生成β-异噁唑-5-酮-丙氨酸(β-(isoxazolin-5-on-2-yl)alanine,BIA),BIA进一步通过两步反应生成β-ODAP。本研究从山黧豆种子萌发6天苗的根中扩增了LsCAS基因的CDS序列,并构建了pGEX2T-LsCAS表达载体。经诱导表达后,通过GST亲和层析进行了LsCAS蛋白纯化,并利用GST标签抗体和大豆半胱氨酸合成酶(Cysteine synthase,CS)抗体对纯化蛋白进行了Western blot验证。纯化后的LsCAS蛋白经凝血酶切除GST标签抗体后,利用凝胶过滤预装柱Superdex 200 Increase 10/300 GL判断分子量表明,LsCAS以四聚体方式存在。在412nm下的特征吸收峰则表明,LsCAS隶属于磷酸吡哆醛(pyridoxal 5"-phosphate,PLP)依赖的半胱氨酸合成酶家族。上述结果为LsCAS酶活调控的理解及进一步的功能研究奠定了坚实的基础。
中文关键词:山黧豆  β-腈基丙胺酸合成酶  原核表达  蛋白纯化  磷酸吡哆醛
 
Prokaryotic Expression and Purification of β-cyanoalanine Synthase in Lathyrus sativus
Abstract:Neuroactive β-N-oxalyl-L-α,β-diaminopropionic acid(β-ODAP)in Lathyrus sativus is neuroprotective at low concentration, while neurotoxic at high concentration to cause lathyrism. Studies on metabolomics and transcriptomics have showed that the biosynthesis of β-ODAP was closely related to basic metabolic pathways such as sulfur metabolism. Isoxazole-5-one and cysteine generates β-(isoxazolin-5-on-2-yl) alanine (BIA) through the catalysis of β-cyanoalanine synthase (β-CAS), by which β-ODAP was further formed by a two-step reaction. So, CDS sequence of LsCAS gene was amplified from the roots of seedlings germinated for 6 days of L, sativus, and pGEX2T-LsCAS expression vector was constructed. LsCAS was purified by GST affinity chromatography after induced expression and detected by western blotting with GST tag antibody and soybean cysteine synthase antibody. The purified LsCAS was digested by thrombin to remove the GST-tag and then molecular weight was estimated by Superdex 200 Increase 10/300 GL. The results showed that LsCAS is tetramer and belongs to pyridoxal phosphate-dependent cysteine synthetase family. These results laid a solid foundation for the understanding of LsCAS activity regulation and further functional investigation.
keywords:Lathyrus  β-ODAP  prokaryotic expression  protein purification  phosphopyridoxal
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