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文章摘要
人来源α-1,3/1,6甘露糖转移酶Alg2的异源表达及活性测定
Heterologous Expression and Activity Assay of Human α-1,3/1,6 Mannosyltransferase Alg2
投稿时间:2019-09-23  修订日期:2019-10-16
DOI:
中文关键词: N-糖基化,Alg2蛋白,甘露糖基转移酶,液质联用
英文关键词: N-glycosylation, Alg2 protein, Mannosyltransferase, LC-MS
基金项目:国家自然科学基金(31971216)
作者单位E-mail
胥欣欣 江南大学 123560215@qq.com 
王春迪 江南大学  
陈帅 江南大学  
高晓冬 江南大学 xdgao@jiangnan.edu.cn 
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中文摘要:
      在N-糖基化途径中,双功能型的甘露糖转移酶Alg2蛋白催化两个甘露糖分别以α-1,3/1,6连键组装到多萜醇寡糖Man1GlcNAc2-PP-Dol上,形成Man3GlcNAc2-PP-Dol。本文旨在探究人来源的Alg2蛋白(hAlg2)在酿酒酵母中的体内功能和在大肠杆菌中表达并纯化后的体外活性。构建酿酒酵母w303a-GAL1pr-ALG2,并利用该菌株的生长被葡萄糖抑制的特点,发现过表达hALG2基因能够回补其生长缺陷,证实了hALG2和ScALG2的功能同源性。本研究利用大肠杆菌表达系统,成功表达并纯化了TrxA-hAlg2,并以Man1GlcNAc2-PP-Phy (PPGn2M1)代替hAlg2的天然底物,测定了其体外活性。结合液质联用的方法,检测发现TrxA-hAlg2具有催化PPGn2M1生成PPGn2M2和PPGn2M3的甘露糖基转移酶活性,为建立基于大肠杆菌表达纯化的hAlg2体外反应体系奠定基础。
英文摘要:
      In the N-glycosylation pathway, the bifunctional mannosyltransferase Alg2 protein catalyzes the assembly of two mannose onto dolichol linked oligosaccharide Man1GlcNAc2-PP-Dol by α-1,3/1,6 linkages, forming Man3GlcNAc2-PP-Dol. In this study, we focused on human Alg2 protein (hAlg2) and intended to explore its function in S. cerevisiae cells and activity in vitro after expressed and purified from E. coli. The growth of constructed yeast strain w303a-GAL1pr-ALG2 was suppressed by glucose, while the overexpression of hALG2 gene can rescue such growth defect, confirming the functional homology of hALG2 and ScALG2. In addition, TrxA-hAlg2 was successfully purified from E. coli and its activity was further tested using PPGn2M1, a natural substrate analogue of hAlg2. LC-MS analysis showed that the purified TrxA-hAlg2 was capable of producing PPGn2M2 and PPGn2M3 from PPGn2M1, providing the foundation for in vitro reaction system of hAlg2 purified from E. coli.
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