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.pdfVIP

现代食品科技 Modern Food Science and Technology 2011, Vol.27, No.4 植物甜蛋白Mabinlin II在大肠杆菌中的表达 王昌博，黄东彦，胡文锋，罗文华，李雪玲，颜少帆，陈永泉 （华南农业大学食品学院生物工程系应用微生物研究室，广东广州 510642） 摘要：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。 关键词：甜味蛋白；马槟榔；Mabinlin II；大肠杆菌；诱导表达 文章篇号：1673-9078(2011)4-384-389 Expression of Plant Sweet Protein Mabinlin II in Escherichia coli WANG Chang-bo, HUANG Dong-yan, HU Wen-feng LUO Wen-hua, LI Xue-ling, YAN Shao-fan, CHEN Yong-quan (Lab of Applied Microbiology, Department of Bioengineering, College of Food Science, South China Agricultural University, Guangzhou 510642, China) Abstract: 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, and the results indicated R-M1 yielded the recombinant Mabinlin II in highest level, and sweetnes