Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2010, Vol. 30 Issue (05): 63-68    DOI: S188
研究报告     
重组玉米f型和m型硫氧还蛋白的功能确定及其靶向蛋白的捕获
冯爱花,张国明,亓振国,范军**
安徽省作物生物学重点实验室 安徽农业大学生命科学学院 合肥 230036
The functional determination of recombinant maize f and m type thioredoxin and entrapment of their target proteinsand entrapment of their target proteinsand entrapment of their target proteins
FENG Ai-hua,ZHANG Guo-ming,Qi Zhen-guo,FAN Jun
The Provincial Key Lab of Crop Science, School of Life Science, Anhui Agricultural University, Hefei 230036, China
 全文: PDF(629 KB)   HTML
摘要:

RT-PCR从玉米幼叶总RNA中克隆f型和m型硫氧还蛋白(Thioredoxin, Trx)的编码基因,分别将两种类型Trx活性中心的第二个保守Cys残基定点突变成Ser残基和Ala残基。在大肠杆菌分别重组表达和纯化了含组氨酸标签的Trx及其突变体蛋白,SDS-PAGE显示纯化的蛋白显示一条主带,蛋白分子量分别估计为f型Trx为18kDa,m型Trx为14kDa;纯化的含有SUMO标签融合Trx,用SUMO专一性SUMO水解酶Ulp除去SUMO,等点聚焦电泳显示m型和f型Trx的等电点分别为4.6和5.9。m型Trx比f型Trx有更强的还原胰岛素能力,而突变体蛋白几乎没有还原能力。用Cys残基专一性标记化合物AMS标记Trx,显示野生型Trx有氧化还原态,而突变体蛋白仅有还原态。SDS-PAGE电泳显示固定化的f型Trx突变体比m型Trx突变体捕获的玉米幼叶靶蛋白更具有多样性。

Abstract:

The genes encoding the mature both types of thioredoxins, f and m type, were cloned by RT-PCR respectively using the total RNA from maize young leaves. The second conservative cystine residues in the catalytic site from two types of the proteins were mutated into serine and alanine residue respectively. The wild type and mutated thioredoxins with histidine-tag were overexpressed in Eecherchia coli and purified. All of them displayed one band on SDS-PAGE. The molecular weight was estimated 18 kD for f type thioredoxin and 14 kD for m type thioredoxin. Both thioredoxins with SUMO fusion tags were also purified and the tags were removed with the SUMO protease Ulp. The proteins displayed pI values of 4.6 for Trx-m and 5.9 for Trx-f. The reduction of insulin suggested that the m type thioredoxin has more active than f type thioredoxin. Both mutated proteins hardly reduced insulin. The modification of the proteins by the specific cystine reagent AMS revealed that the purified wild type thioredoxins displayed the redox states, but the mutated thioredoxins showed the reduced state, suggesting that there is no disulfide bond in the mutated thioredoxins. The entrapped proteins from young leaves of maize by the mutated f type thioredoxin with histidine-tag immobilized on the Ni-NTA resin were more diverse than those by the mutated m type thioredoxin, as shown by SDS-PAGE.

收稿日期: 2010-01-21 出版日期: 2010-05-25
基金资助:

国家自然科学基金(30840018)资助项目

通讯作者: 范军     E-mail: fanjun@ahau.edu.cn
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
冯爱花
张国明
亓振国
范军

引用本文:

冯爱花 张国明 亓振国 范军. 重组玉米f型和m型硫氧还蛋白的功能确定及其靶向蛋白的捕获[J]. 中国生物工程杂志, 2010, 30(05): 63-68.

FENG Ai-Hua, ZHANG Guo-Meng, QI Zhen-Guo, FAN Jun. The functional determination of recombinant maize f and m type thioredoxin and entrapment of their target proteinsand entrapment of their target proteinsand entrapment of their target proteins. China Biotechnology, 2010, 30(05): 63-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/S188        https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I05/63

[1] Schürmann P, Buchanan B B. The ferredoxin/thioredoxin system of oxygenic photosynthesis. Antioxid Redox Signal, 2008, 10(7): 12351274. 
[2] Lemaire S D, Michelet L, Zaffagnini M, et al. Thioredoxins in chloroplasts. Curr Genet, 2007, 51(6): 343365. 
[3] Meyer Y, Reichheld J P, Vignols F. Thioredoxins in Arabidopsis and other plants. Photosynth Res, 2005, 86(3): 419433. 
[4] Barajas J, Serrato A J, Olmedilla A, et al. Localization in roots and flowers of pea chloroplastic thioredoxin f and thioredoxin m proteins reveals new roles in nonphotosynthetic organs. Plant Physiol, 2007, 145(3): 946960. 
[5] Motohashi K, Kondoh A, Stump P M T, et al. Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci U S A, 2001, 98(20): 1122411229. 
[6] Balmer Y, Koller A, Val G D, et al. Proteomics uncovers proteins interacting electrostatically with thioredoxin in chloroplasts. Photosynth Res, 2004, 79(3): 275280. 
[7] Hall M, Cabana A, Akerlund H E, et al. Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function. Proteomics, 2010, 10(5): 9871001. 
[8] Nuruzzaman M, Gupta M, Zhang C, et al. Sequence and expression analysis of the thioredoxin protein gene family in rice. Mol Genet Genomics, 2008, 280(2): 139151. 
[9] Chi Y H, Moon J C, Park J H, et al. Abnormal chloroplast development and growth inhibition in Oryza sativa thioredoxin m knockdown plants. Plant Physiol, 2008, 148(2): 808817. 
[10] Alexandrov N N, Brover V V, Freidin S, et al. Insights into corn genes derived from largescale cDNA sequencing. Plant Mol Biol, 2009, 69(12): 179194. 
[11] Malakhov M P, Mattern M R, Malakhova O A, et al. SUMO fusions and SUMOspecific protease for efficient expression and purification of proteins. J Struct Funct Genomics, 2004, 5(12): 7586. 
[12] Maeda K, Finnie C, Stergaard O, et al. Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome. Eur J Biochem, 2003, 270(12): 26332643. 
[13] Ikegami A, Yoshimura N, Motohashi K, et al. The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J Biol Chem, 2007, 282(27): 1928219291. 
[14] Rey P, Cuiné S, Eymery F, et al. Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses. Plant J, 2005, 41(1): 3142. 
[15] Cazalis R, Pulido P, Aussenac T, et al. Cloning and characterization of three thioredoxin h isoforms from wheat showing differential expression in seeds. J Exp Bot, 2006, 57(10): 21652172. 
[16] Capitani G, Housley Z, DelVal G, et al. Crystal structures of two functionally different thioredoxins in spinach chloroplasts. J Mol Biol, 2000, 302(1): 135154. 
[17] Lamotte F, Miginiac M, Decottignies P, et al. Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes. Eur J Biochem, 1991, 196(2): 287294. 
[18] Maeda K, Hgglund P, Finnie C, et al. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin. Structure, 2006, 14(11): 17011710. 
[19] Saze H, Ueno Y, Hisabori T, et al. Thioredoxinmediated reductive activation of a protein kinase for the regulatory phosphorylation of C4form phosphoenolpyruvate carboxylase from maize. Plant Cell Physiol, 2001, 42(12): 12951302. 
[20] Kleczkowski L A, Randall D D. Light and thiol activation of maize leaf glycerate kinase: the stimulating effect of reduced thioredoxins and ATP. Plant Physiol, 1985, 79(1): 274277.

No related articles found!