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Role of the CPC Sequence in the Antioxidant Activity of GcGAST Protein in E.coli.
MedLine Citation:
PMID:  23300060     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine-Proline-Cysteine (SPC) and Cysteine-Proline-Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine-Proline-Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H(2)O(2). Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H(2)O(2). Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H(2)O(2), whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H(2)O(2)-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more β-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H(2)O(2). More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.
Authors:
Ying-Nan Bai; Juan Feng; Hong-Ju Ma; Jun-Yue Lin; Shao-Bo Han; Li-Xia Tang
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-9
Journal Detail:
Title:  The protein journal     Volume:  -     ISSN:  1875-8355     ISO Abbreviation:  Protein J.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-9     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101212092     Medline TA:  Protein J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
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