We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Novel Method for the Production, Purification, and Characterization of Recombinant Lunasin: Identification of Disulfide Cross-Linked Dimers.
- Authors
Alves de Souza, Stephanny Miranda; de Araújo, Talita Stelling; Ferretti, Giulia Diniz da Silva; Kalume, Dário Eluan; Cordeiro, Yraima; Almeida, Marcius da Silva; de Souza, Theo Luiz Ferraz
- Abstract
Lunasin is a soybean peptide with promising therapeutic applications in cancer and other diseases. It is described as an intrinsically disordered peptide, monomeric, and that can form an intramolecular disulfide bond between Cys10 and Cys22. In this study, we tested a new approach to obtain recombinant lunasin by Escherichia coli expression and performed its structural characterization. We expressed a lunasin sequence with an N-terminal 6× His-tag, B1 domain of Streptococcal protein G (GB1), and Tobacco etch virus (TEV) cleavage site (His6-GB1-lunasin), using pET-25b(+) vector. His6-GB1-lunasin purification using immobilized metal affinity chromatography achieved high recovery. We obtained a final yield of 12.0 (± 0.39) mg/L of recombinant lunasin with high purity. The molecular mass and conformation were as expected, as verified by liquid chromatography–mass spectrometry (LC–MS), electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS), electrospray ionization–mass spectrometry (ESI–MS), size-exclusion chromatography, circular dichroism, fluorescence spectroscopy, and one-dimensional 1H nuclear magnetic resonance. Additionally, the identity of lunasin and its complete amino acid sequence was confirmed by LC–MS/MS. Recombinant lunasin also showed antioxidant activity (2.92 ± 0.16 µmol of trolox equivalents/µmol lunasin) by oxygen radical absorbance capacity and inhibited cell migration of MDA-MB-231 cell line. On the other hand, for the first time, LC–MS, ESI–IMS–MS and ESI–MS analyses revealed, in addition to the reduced and oxidized monomeric forms, the presence of small populations of disulfide cross-linked dimeric species of lunasin. Overall, our data demonstrate the effectiveness of the GB1-tagging system to obtain lunasin, that lunasin can form disulfide cross-linked dimers, and that LC–MS, ESI–IMS–MS and ESI–MS are efficient analytical chemistry techniques to assess reduced and oxidized (monomer and dimer) species.
- Publication
International Journal of Peptide Research & Therapeutics, 2022, Vol 28, Issue 6, p1
- ISSN
1573-3149
- Publication type
Article
- DOI
10.1007/s10989-022-10466-2