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0.05–0.01), compared to control results, regarding LPO, SOD, GST and CAT. In case of AChE, supplementation of zinc showed little alteration in the activity of this enzyme in the rats treated with chlorpyrifos. It can deduce that chlorpyrifos induced oxidative stress and lipid peroxidation in erythrocytes of male and female rats. The overall results reveal the pronounced ameliorating effect of zinc in chlorpyrifos-intoxicated rats and variation in the response of male and female animals regarding alteration in the level of some biochemical parameters and LPO.
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doi:10.1016/j.pep.2006.09.007 
Copyright © 2006 Elsevier Inc. All rights reserved.
Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system
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Takuya Kannoa, Michiko Kitanob, Rika Katob, Akira Omorib, Yaeta Endoa, c and Yuzuru Tozawaa, c, 
, 
Received 8 July 2006;
Abstract
Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (
10 μg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.
Keywords: Cell-free protein synthesis; Wheat-embryo; N-terminal processing; Methionine aminopeptidase
Article Outline
- Materials and methods
- Cell-free protein synthesis and protein purification
- N-terminal amino acid sequence analysis
- Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- Results
- Synthesis of MalE- and GST-variants
- Sequence analysis for synthesized proteins
- Effect of antepenultimate amino acid residue on N-Met cleavage
- Mass spectrometric analysis of MalE mutants
- Comparison of N-Met cleavage efficiency between two different protein synthesis-methods, dialysis method and the bilayer translation method
- Post-translational N-terminal processing by intrinsic MAP in wheat-embryo extract
- Discussion
- Acknowledgements
- References
