天津科技大学学报2024，Vol.39Issue(4)：46-53,8.DOI:10.13364/j.issn.1672-6510.20230114
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
摘要
Abstract
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 sulfotrans-ferases 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 synthe-sized into the expression vector pET-32a,and the E.coli BL21 was transferred to induce expression.The heterologous ex-pressions 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 fur-ther 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Key words
sulfotransferases/3′-pho-sphoadenosine-5′-phosphosulfate(PAPS)/heparin/Escherichia coli/expression分类
生物科学引用本文复制引用
龚巧琳,杨书尧,寇梦云,宋亚囝,郑迎迎,李金山..新型芳基磺基转移酶在大肠杆菌中的表达及表征[J].天津科技大学学报,2024,39(4):46-53,8.基金项目
国家重点研发计划项目(2021YFC2103200) （2021YFC2103200）
天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-009) （TSBICIP-KJGG-009）
