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      Expression of Bioactive Lunasin Peptide in Transgenic Rice Grains for the Application in Functional Food

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          Abstract

          Lunasin, a bioactive peptide initially isolated from soybean, has anticancer, anti-inflammatory, and antioxidant activity. Due its great application value, lunasin seems to be a candidate gene in improving the nutritional value of crops. In this study, lunasin was inserted into the rice genome to evaluate whether it was feasible to express lunasin using the rice expression system and improve the bioactivity of protein in rice for our needs. We generatedlunasin-overexpressing rice lines, and chose three independent transgenic rice lines for further study. The lunasin content in trans-lunasin rice detected by UPLC-MS/MS was 1.01 × 10 −3 g·kg −1 dry rice flour with grease removal in the lunasin extracts. The antioxidant efficacy of LET (lunasin-enriched fraction from trans-lunasin rice) and PEW (peptide-enriched fraction from wild type rice) was compared. Due to the presence of lunasin, LET showed higher ( p < 0.05) antioxidant activity than PEW. LET exhibited high DPPH radical scavenging activity (IC 50 value, 8 g·L −1), strong ABTS + radical scavenging activity (IC 50 value, 1.18 g·L −1), and great oxygen radical scavenging activity (170 μmol·L −1 Trolox equivalents when the concentration reached 4 g·L −1). Moreover, LET presented significantly higher ( p < 0.05) anti-inflammatory activity on macrophage cells, and the NO production and the release of pro-inflammatory cytokines (IL-6, MCP1, and TNF-α) were significantly inhibited by LET. However, because of the low purity, LET showed weaker antioxidant and anti-inflammatory activity when compared to the Lunasin standard. These results suggested that it is feasible to use the rice expression system to express the exogenous lunasin in rice, and lunasin-overexpressing rice seems to be a candidate resource for application in functional food. Rice rich in lunasin is beneficial for human health, and could be used as a functional food in the diets of cancer and obese patients in the future.

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          Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts

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            Western blot: technique, theory, and trouble shooting.

            Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.
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              Recombinant expression systems in the pharmaceutical industry.

              F. Schmidt (2004)
              In terms of downstream processing efficiency, secretory expression systems offer potential advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems. However, for high-volume therapeutics like insulin, the product yields of the majority of the potentially available secretory systems is not yet fully competitive. Current strategies to improve productivity and secretion efficiency comprise: (1) enhancement of gene expression rates, (2) optimization of secretion signal sequences, (3) coexpression of chaperones and foldases, (4) creation of protease deficient mutants to avoid premature product degradation and (5) subsequent breeding and mutagenesis. For the production of non-glycosylated proteins and proteins, which are natively glycosylated but are also pharmacologically active without glycosylation, prokaryotes, which usually lack metabolic pathways for glycosylation, are theoretically the most suitable organisms and offer two alternatives: either Escherichia coli strains are conditioned to be efficient secreters or efficient native secreters like Bacillus species are accordingly developed. To fully exploit the secretory capacity of fungal species, a deeper understanding of their posttranslational modification physiology will be necessary to steer the degree and pattern of glycosylation, which influences both folding and secretion efficiency. Insect and mammalian cells display posttranslational modification patterns very similar or identical to humans, but in view of the entailed expenditures, their employment can only be justified if their modification machinery is required to ensure a desired pharmacological activity.
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                Author and article information

                Journal
                Molecules
                Molecules
                molecules
                Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry
                MDPI
                1420-3049
                17 September 2018
                September 2018
                : 23
                : 9
                : 2373
                Affiliations
                [1 ]College of Pharmacy and Biological Engineering, Chengdu University, No. 1 Shilling Road, Chenglo Avenue, Longquan District, Chengdu 610106, China
                [2 ]Institute of Crop Science, Chinese Academy of Agricultural Sciences, No. 80 South Xueyuan Road, Haidian, Beijing 100081, China; haoyuqiong334@ 123456163.com (Y.H.); zhuying881020@ 123456163.com (Y.Z.); shizhenxing@ 123456caas.cn (Z.S.)
                [3 ]Precision Livestock and Nutrition Unit, Gembloux Agro-Bio Tech, TERRA Teaching and Research Centre, University of Liège, Passage des Déportés, 5030 Gembloux, Belgium
                [4 ]Laboratory of Biomass and Green Technologies, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés, 5030 Gembloux, Belgium
                [5 ]Key Laboratory of Coarse Cereal Processing, Ministry of Agriculture, Chengdu University, No.1 Shilling Road, Chenglo Avenue, Longquan District, Chengdu 610106, China
                Author notes
                [* ]Correspondence: renguixing@ 123456cdu.edu.cn (G.R.); zhaogang@ 123456cdu.edu.cn (G.Z.); Tel.: +86-10-6211-5596 (G.R.); Fax: +86-10-6215-6596 (G.R.)
                [†]

                These authors have contributed equally to this work.

                Article
                molecules-23-02373
                10.3390/molecules23092373
                6225134
                30227638
                89fb9153-d8f1-4ee3-adce-ab54279e0962
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 07 August 2018
                : 04 September 2018
                Categories
                Article

                lunasin,transgenic rice,uplc-ms/ms,antioxidant activity,anti-inflammatory activity

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