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Functional Research of Soybean Bioactive Peptide -Prokaryotic Expression, Purification and Bioassay of Lunasin

Author: GaiWenLi
Tutor: ChenZhengWang
School: Huazhong University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Lunasin Prokaryotic Expression Vector Construction Affinity Chromatography Western Blot Bioassay
CLC: Q946.1
Type: Master's thesis
Year: 2010
Downloads: 98
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Abstract

Lunasin is a 43 amino acid residues polypeptide with a mass of 5kDa,characterd as a soybean bioactive peptide by a Japanese research team in the 1987. Lunasin contains the cell adhesion motif Arg-Gly-Asp(RGD), known to allow attachment to the extracellular matrix. It also contains 8 Asp residues in its carboxyl end that have been found to be responsible for its antimitotic effect, after their binding to regions of hypoacetylated chromatin (such as in centromeres), leading to mitotic arrest and eventually to cell death. It has been demonstrated that Lunasin can prevent colon cancer, breast cancer and prostate cancer. It can also inhibite in?ammation by suppressing NF-κB pathway in LPS-induced RAW264.7 macrophage. For the interesting structure and function of Lunasin, it is very important for us to study the bioactive peptide Lunasin.Lunasin content is too low to purify from natural materials, purification is complicated and expensive. So the gene of Lunasin was artificially synthetic based on the amino-acid sequence of Lunasin. After enzyme hydrolysis, conjunction, the fragment was cloned into prokaryotic expression vector pET-32a(+). The recombinant expression vector pET-32a(+)-Lunasin-6His was transformed into E.coli BL21(DE3). The conditions of inducement were optimized at 30℃, 0.05mM IPTG, 4h. The fusion protein Lunasin was solubly expressed. The molecular mass of Lunasin-6His was 6kDa. The fusion protein was identified by SDS-PAGE and Western Blot. Bioassay of recombinant Lunasin was measured by MTT and NO detection.The work provides preparation for further study of the function of Lunasin and producing the protein with gene engineering for clinical application.

Functional Research of Soybean Bioactive Peptide -Prokaryotic Expression, Purification and Bioassay of Lunasin

Abstract

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