植物甜蛋白Mabinlin Ⅱ在大肠杆菌中的表达

Expression of Plant Sweet Protein Mabinlin II in Escherichia coli

本研究从马槟榔种子中扩增得到天然植物甜蛋白Mabinlin Ⅱ的全长cDNA序列(M1),并通过特异引物PCR去除部分5’端序列得到修饰型的基因序列(M2)。将M1与M2分别克隆至表达载体pET43.1a(+),并分别转化表达宿主菌Escherichia coli BL21(DE3)和Rosetta(DE3),成功构建出BL21(DE3)/pET43.1a-M1(缩写B-M1)、BL21(DE3)/pET43.1a-M2(缩写B-M2)、Rosetta(DE3)/pET43.1a-M1(缩写R-M1)、Rosetta(DE3)/pET43.1a-M2(缩写R-M2)四个表达系统。经IPTG诱导表达后证明,R-M1的重组甜蛋白表达量最高,并可检测到轻微甜味和后甜感。同时利用镍柱对R-M1诱导表达的目的蛋白进行纯化,得到单一条带的重组甜蛋白。

Mabinlin Ⅱ is a safe,low calorie and strong sweet protein found in the seed of Capparis masaikai Levl grown in Yunnan Province Southern China.We amplified the whole cDNA sequence of Mabinlin II（M1） and the modified gene sequence（M2）,which without 105bp of the 5＇terminal from the seeds,and cloned into the expression plasmid pET43.1a（＋） respectively.The two recombinant plasmid were then transferred into Escherichia coli BL21（DE3） and Rosetta（DE3）,resulting in 4 recombinant expression system：BL21（DE3）/pET43.1a-M1（abbr：B-M1）,BL21（DE3）/pET43.1a-M2（abbr：B-M2）,Rosetta（DE3）/pET43.1a-M1（abbr：R-M1） and Rosetta/pET43.1a-M2（abbr：R-M2）.Recombinant proteins expression was detected by SDS-PAGE,and the results indicated R-M1 yielded the recombinant Mabinlin II in highest level,and sweetness was also detected.The R-M1 recombinant protein was then purified using Ni-NTA resin,and single protein band was observed after SDS-PAGE.