新型芳基磺基转移酶在大肠杆菌中的表达及表征  

Expression and Characterization of Novel Sulfotransferases in Escherichia coli

作　　者：龚巧琳 杨书尧 寇梦云 宋亚囝 郑迎迎 李金山 GONG Qiaolin;YANG Shuyao;KOU Mengyun;SONG Yajian;ZHENG Yingying;LI Jinshan(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《天津科技大学学报》2024年第4期46-53,共8页Journal of Tianjin University of Science & Technology

基　　金：国家重点研发计划项目(2021YFC2103200);天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-009)。

摘　　要：肝素是动物源高度硫酸化、非均一的线性糖胺聚糖。芳基磺基转移酶参与肝素生物合成中磺基供体3′–磷酸腺苷–5′–磷酰硫酸(PAPS)的合成与再生,是实现肝素生理功能的关键酶。目前已报道的芳基磺基转移酶存在种类少、表达量低等问题。本研究采用隐马尔可夫模型挖掘获得10个鸟类来源的磺基转移酶,对新基因进行密码子优化并全合成到表达载体pET-32a上,转入大肠杆菌(Escherichia coli)BL21进行诱导表达。通过优化培养基成分、诱导温度、异丙基硫代-β-D-半乳糖苷(IPTG)浓度以及摇床转速,成功实现S4和S8的异源表达。在TB培养基中、诱导温度为16℃、使用0.4 mmol/L IPTG进行诱导、200 r/min转速条件下,菌株BL21/pET32a-Mbp-S4的S4蛋白表达量为60.6 mg/L。对S4蛋白进行纯化和酶活力表征,S4蛋白的最适反应温度为40℃,最适反应pH为7.0,比活力为3.3 mU/mg。通过超高效液相色谱-质谱(UPLC-MS)检测酶修饰后产物,进一步验证S4蛋白为芳基磺基转移酶。芳基磺基转移酶S4的成功表达为PAPS再生提供了功能元件。Heparin is a highly sulfated and heterogeneous linear glycosaminoglycan of animal origin.Aryl sulfotransferase participates in the synthesis and regeneration of 3′-pho-sphoadenosine-5′-phosphosulfate(PAPS)of the sulfonic acid donor in heparin biosynthesis,and is a key enzyme to realize the physiological function of heparin.At present,aryl sulfotransferases have been reported to have such problems as few species and low expression amount.In our present study,the hidden Markov model was used to obtain 10 sulfotransferases derived from birds,and the codon was optimized and fully synthesized into the expression vector pET-32a,and the E.coli BL21 was transferred to induce expression.The heterologous expressions of S4 and S8 were achieved successfully by optimizing the medium composition,induction temperature,isopropylthio-β-D-galactoside(IPTG)concentration and shaker speed.In TB medium,induction at 16℃using 0.4 mmol/L IPTG was expressed with strain BL21/pET32a-Mbp-S4 at 200 r/min.The expression of the target protein reached 60.6 mg/L.The purification and characterization of S4 proteins showed that the optimal reaction temperature,pH and specific enzyme activity reached 40℃,7.0 and 3.3 mU/mg,respectively.The modified product was detected by UPLC-MS,and S4 was further verified as sulfonyltransferase.The successful expression of novel sulfotransferases S4 has provided an effective element for PAPS regeneration.

关 键 词：磺基转移酶 3′–磷酸腺苷–5′–磷酰硫酸(PAPS) 肝素 大肠杆菌 表达 

分 类 号：Q71[生物学—分子生物学]