摘要
【背景】目前利用酵母表达系统已鉴定了多种物种中的Δ6脂肪酸脱饱和酶(FADS6)。由于FADS6是一种具有多个跨膜螺旋的膜蛋白,使得其大量表达和纯化具有挑战性。【目的】探索FADS6的高效表达策略,研究纯化标签添加的位置对高山被孢霉FADS6I(Ma FADS6I)重组表达效率的影响。【方法】在毕赤酵母表达载体中插入串联亲和标签HRV 3C-Protein A-His,利用改造后的载体构建带有N端或C端标签的Ma FADS6I表达载体;通过电转化获得毕赤酵母重组表达菌株;利用斑点印迹杂交(DotBlot)、聚丙烯酰胺凝胶电泳(SDS-PolyacrylamideGelElectrophoresis,SDS-PAGE)和免疫印迹(Western Blot)分析重组蛋白的表达水平,并利用气相色谱-质谱(Gas Chromatography-Mass Spectrometry,GC-MS)分析检测Ma FADS6I催化生成的脂肪酸。【结果】通过大量的毕赤酵母转化子筛选,最终获得高效表达Ma FADS6I的毕赤酵母重组菌,证实各转化子的表达具有差异性,Ma FADS6I的C端带有纯化标签较N端更有利于表达。【结论】在Ma FADS6I的C端添加纯化标签比在N端添加更有利于该蛋白在酵母系统中的表达以及底物的转化,为进一步探究FADS6高效表达和结构功能奠定了基础。
[Background]At present,the characteristics ofΔ6 fatty acid desaturase(FADS6)from various species have been identified through the yeast expression system.Since FADS6 is a multiple transmembrane protein,it is challenging to achieve large-scale expression and purification.[Objective]To construct a high-efficiency expression strategy of FADS6,the present study will analyze the influence of the location of the purification tag on heterologous expression of Mortierella alpina FADS6 I(Ma FADS6 I).[Methods]Tandem affinity tag HRV 3 C-Protein A-His was added into the Pichia pastoris vector,followed by the insertion of Ma FADS6 I sequence to construct recombinant vectors with the N-terminal or C-terminal tag,respectively.Recombinants were obtained through electro-transformation.The protein expression level of Ma FADS6 I in recombinant strains was analyzed by dot blot hybridization(dot blot),polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot,and the fatty acids catalyzed by Ma FADS6 I was detected by gas chromatography-mass spectrometry(GC-MS).[Results]Transformants with different Ma FADS6 I expression levels and catalytic activities were obtained.Compared with the N-terminal tag,the C-terminal tag was more conducive for the expression and catalytic activity of Ma FADS6 I.[Conclusion]Ma FADS6 I with C-terminal purification tag is more conducive to the expression of the protein in the yeast system and the conversion of substrates than with N-terminal tag,providing a foundation for the high-efficiency expression and structural and functional studies of FADS6.
作者
崔洁
陈海琴
唐鑫
张灏
陈永泉
陈卫
CUI Jie;CHEN Haiqin;TANG Xin;ZHANG Hao;CHEN Yongquan;CHEN Wei(School of Food Science and Technology,Jiangnan University,Wuxi,Jiangsu 214122,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2021年第8期2607-2618,共12页
Microbiology China
基金
国家自然科学基金(31722041)。
