Cell-Free Expression, Purification, and Characterization of the Functional beta(2)-Adrenergic Receptor for Multianalyte Detection of beta-Agonists

文献类型： 外文期刊

第一作者： Wang, Jian

作者： Wang, Jian;Liu, Yuan;Han, Zhengzheng;Wang, Wei;Liu, Yang;Wei, Dong;Wang, Jian;Liu, Yuan;Zhang, Junhua;Huang, Wei

作者机构：

关键词： cell-free expression;beta(2)-adrenergic receptor;codon optimization;purification;beta-agonist;receptor-based assay

期刊名称：BIOCHEMISTRY-MOSCOW （ 影响因子：2.487； 五年影响因子：2.57 ）

ISSN： 0006-2979

年卷期： 2017 年 82 卷 11 期

页码：

收录情况： SCI

摘要： Large-scale expression of beta(2)-adrenergic receptor (beta(2)-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused beta-adrenergic agonists (beta-agonists). Cell-based heterologous expression systems have many critical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional beta(2)-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine beta(2)-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified beta(2)-AR for beta-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC50 values were 45.99, 60.38, and 78.02 mu g/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of beta(2)-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting beta-agonist residues.

分类号：

  • 相关文献

[1]Simultaneous Determination of 14 Kinds of beta-Agonist Residues in Sheep Saliva by Ultra Performance Liquid Chramatogrephy-Tandem Mass Spectrometry. Li Yang,Su Xiao-Ou,Wang Rui-Guo,Wang Pei-Long,Zhang Wei,Chen Jie. 2013

[2]A sensitive and quantitative fluorescent multi-component immuno-chromatographic sensor for beta-agonist residues. Wang, Peilong,Su, Xiaoou,Wang, Peilong,Su, Xiaoou,Wang, Zhi.

[3]Direct measurement of beta-agonists in swine hair extract in multiplexed mode by surface-enhanced Raman spectroscopy and microfluidic paper. Dou, Bin,Luo, Yong,Gao, Zhigang,Zhao, Weijie,Lin, Bingcheng,Dou, Bin,Luo, Yong,Gao, Zhigang,Zhao, Weijie,Chen, Xu,Shi, Bo,Du, Yuguang,Lin, Bingcheng.

[4]Non-uniqueness of factors constraint on the codon usage in Bombyx mori. Jia, Xian,Zheng, Hao,Qi, Qi,Wei, Lei,Sun, Jingchen,Jia, Xian,Zheng, Hao,Qi, Qi,Wei, Lei,Sun, Jingchen,Liu, Shuyu,He, Jian,Li, Bo,Zhao, Taiyi. 2015

[5]High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization. Gao, Zhaowei,Zhang, Yuhong,Li, Mu,Zhou, Liwei,Zhang, Wei,Gao, Zhaowei,Tang, Yunming,Li, Zhuofu,Huang, Huoqing,Yao, Bin. 2012

[6]Improved secretory production of calf prochymosin by codon optimization and disruption of PMR1 in Kluyveromyces lactis GG799. Feng, Zhen,Ren, Jing,Zhang, Yanhe,Hu, Sen. 2011

[7]Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Teng, Da,Fan, Ying,Yang, Ya-lin,Tian, Zi-gang,Luo, Jin,Wang, Jian-hua.

[8]Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes. Cao, Yimei,Sun, Pu,Fu, Yuanfang,Bai, Xingwen,Tian, Feipen,Liu, Xiangtao,Lu, Zengjun,Liu, Zaixin.

[9]High level expression of an acid-stable phytase from citrobacter freundii in pichia pastoris. Zhao, Wei,Xiong, Aisheng,Fu, Xiaoyan,Gao, Feng,Tian, Yongsheng,Peng, Rihe.

[10]High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody. Li, Chuanfeng,Chen, Zongyan,Meng, Chunchun,Li, Lu,Liu, Guangqing. 2013

[11]Chloroplast-targeted expression of the codon-optimized truncated cry1Ah gene in transgenic tobacco confers a high level of protection against insects. Huang, Dafang,Li, Xiuying,Li, Shengyan,Lang, Zhihong,Zhu, Li,Huang, Dafang,Li, Shengyan,Zhang, Jie. 2013

[12]Immunogenicity of virus-like particles containing modified goose parvovirus VP2 protein. Chen, Zongyan,Li, Chuanfeng,Zhu, Yingqi,Wang, Binbin,Meng, Chunchun,Liu, Guangqing.

[13]Codon optimization and woodchuck hepatitis virus posttranscriptional regulatory element enhance the immune responses of DNA vaccines against infectious bursal disease virus in chickens. Li, Kai,Gao, Li,Gao, Honglei,Qi, Xiaole,Gao, Yulong,Qin, Liting,Wang, Yongqiang,Wang, Xiaomei.

[14]Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells. Gao, Jingpeng,Meng, Chunchun,Chen, Zongyan,Li, Chuanfeng,Liu, Guangqing.

[15]Overexpression of D-amino acid oxidase from Bradyrhizobium japonicum, enhances resistance to glyphosate in Arabidopsis thaliana. Han, Hongjuan,Zhu, Bo,Fu, Xiaoyan,You, Shuanghong,Wang, Bo,Li, Zhenjun,Zhao, Wei,Peng, Rihe,Yao, Quanhong,You, Shuanghong,Yao, Quanhong.

[16]Protection of chickens against reticuloendotheliosis virus infection by DNA vaccination. Li, Kai,Gao, Li,Gao, Honglei,Qi, Xiaole,Gao, Yulong,Qin, Liting,Wang, Yongqiang,Wang, Xiaomei. 2013

[17]Cloning, codon-optimized expression and homology modeling of structural protein VP1 from foot and mouth disease virus. Liu, Xin-sheng,Wang, Yong-lu,Zhang, Yong-guang,Fang, Yu-zhen,Pan, Li,Lue, Jian-liang,Zhou, Peng,Zhang, Zhong-wang,Cheng Qi-wei,Wang, Gang,Wang, Ji-wei,Lou, Hui,Jiang, Shou-tian. 2011

[18]Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector. Jiang, Yongping,Yu, Kangzhen,Zhang, Hongbo,Zhung, Pingjing,Li, Chenjun,Tian, Guobin,Li, Yanbing,Wang, Xijun,Ge, Jinying,Bu, Zhigao,Chen, Hualan. 2007

[19]Porcine reproductive and respiratory syndrome virus ORF5a protein is essential for virus viability. Lin, Tao,Huang, Ting,Yao, Huochun,Fan, Hongjie,Sun, Lichang,Li, Yanhua,Liu, Runxia,Gao, Fei,Tong, Guangzhi,Wei, Zuzhang,Yuan, Shishan,Li, Yanhua,Wang, Xiaomin.

[20]Extracellular expression and antiviral activity of a bovine interferon-alpha through codon optimization in Pichia pastoris. Tu, Yabin,Wang, Gang,Wang, Yanqun,Chen, Weiye,Zhang, Lu,Liu, Yonggang,Jiang, Chenggang,Wang, Shujie,Bu, Zhigao,Cai, Xuehui.

作者其他论文 更多>>

微信小程序