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- Title
Role of the CPC Sequence in the Antioxidant Activity of GcGAST Protein in E.coli.
- Authors
Bai, Ying-nan; Feng, Juan; Ma, Hong-Ju; Lin, Jun-yue; Han, Shao-bo; Tang, Li-Xia
- Abstract
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 HO. Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to HO. Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate HO, whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of HO-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 HO. More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.
- Subjects
ANTIOXIDANTS; AMINO acid sequence; CYSTEINE; PLANT proteins; PROLINE; ESCHERICHIA coli; ORCHIDS; GIBBERELLIC acid
- Publication
Protein Journal, 2013, Vol 32, Issue 1, p48
- ISSN
1572-3887
- Publication type
Article
- DOI
10.1007/s10930-012-9460-3