Structure and Function of Heat-stable Sweet Protein, Mabinlin

热稳定甜味蛋白马宾林的结构和功能

基本信息

批准号：
05044126
负责人：
KURIHARA Yoshie
金额：
$ 3.84万
依托单位：
Yokohama National University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05044126/
关键词：
Sweet protein heat-stable protein mabinlin cDNA cloning

项目摘要

Total RNA was extracted by the phenol-SDS method from the seeds of Capparis masaikai Levl. Poly (A) ^+RNA was purified by Oligotex-dT30 Super. Double-stranded cDNA was synthesized using a cDNA synthesis kit. lambdagt10 cDNA library was constructed with Complete rapid cloning system-lambdagt10. Two oligonucleotide primers, 5'-AGCATATGCA (AG) (TC) TITGG (CA) GITG (TC) CA-3' and 5'-CGGATCCCTACCAIGCIC (GT) AGAAIGG (AG) C-3', were prepared on the basis of the amino acid sequences QLWRCQ and CPFRAW,respectively. The PCR was conducted using the cDNA from seeds with 30 cycles of 1 min at 96 ﾟC for denaturation, 1 min at 50ﾟC for annealing, and 1 min at 72ﾟC for extension. The amplified DNA fragment was used as a probe to screen the cDNA library. Recombinant phages were screened by plaque hybridization with the ^<32>P-labeled probe. We finally obtained 5 clones out of 1.0x10^5 plaques. One clone with a cDNA insert of 630 bp was subjected to nucleotide sequencing. The clone contained single open reading frame of 465 bp. The amino acid sequence determined in a previous study matched the deduced sequence from 36 to 68 and from 85 to 155. The N-terminal extension of 20 amino acids (Met^1 to Ala^<20>) is rich in hydrophobic amino acid and appears to be a signal sequence.The DNA encoding mabinlin II was inserted into the expression vector, pET15b. This expression vector termed pMAB2 contained bacteriophage T7 promoter. The vector was used to transform E.coli strain BL21. E.coli BL21/pMAB2 was induced by isopropylthio-beta-D-galactoside (IPTG) in LB medium. The protein with an apparent molecular weight (15kDa) identical to that of mabinlin II protein was detected by SDS-PAGE of whole cell lysates. Western blotting analysis showed that this induced protein reacted with anti-mabinlin II antibody.

通过苯酚-SDS方法从卡帕里斯Masaikai Levl的种子中提取总RNA。 poly（a） ^+RNA通过oligotex-dt30 super纯化。使用cDNA合成试剂盒合成双链cDNA。 Lambdagt10 cDNA库是由完全快速克隆系统lambdagt10构建的。 Two oligonucleotide primers, 5'-AGCATATGCA (AG) (TC) TITGG (CA) GITG (TC) CA-3' and 5'-CGGATCCCTACCAIGCIC (GT) AGAAIGG (AG) C-3', were prepared on the basis of the amino acid sequences QLWRCQ and CPFRAW, respectively.使用来自96°C的30分钟循环的种子的cDNA进行变性，在50°C进行1分钟进行退火，并在72℃下进行1分钟进行PCR进行。放大的DNA片段用作筛选cDNA文库的探针。通过与 ^<32> p标记的探针通过斑块杂交筛选重组噬菌体。我们终于在1.0x10^5斑块中获得了5个克隆。一个带有630 bp的cDNA插入物的克隆进行核测序。克隆包含465 bp的单个开放式阅读框。在先前的研究中确定的氨基酸序列匹配了36至68的推导序列，从85到155。20个氨基酸的N末端扩展（Met^1至Ala^<20>）富含疏水性氨基酸，似乎是信号序列。DNA编码Mabinlin II的DNA插入了表达矢量，PET15B。该称为pMAB2的表达载体包含细菌噬菌体T7启动子。该载体用于转化大肠杆菌菌株BL21。在LB培养基中，通过异丙基甲基二-D-半乳糖苷（IPTG）诱导大肠杆菌BL21/PMAB2。通过全细胞裂解物的SDS-PAGE检测到与Mabinlin II蛋白相同的蛋白质（15KDA）与Mabinlin II蛋白相同的蛋白质。蛋白质印迹分析表明，该诱导的蛋白与抗马比林II抗体反应。