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中国生物工程杂志

China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (05): 63-68    DOI: S188
    
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
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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.



Received: 21 January 2010      Published: 25 May 2010
Cite this article:

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.

URL:

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

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