Novel Method for the Production, Purification, and Characterization of Recombinant Lunasin: Identification of Disulfide Cross-Linked Dimers

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 Cys(10) and Cys(22). 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 6x His-tag, B1 domain of Streptococcal protein G (GB1), and Tobacco etch virus (TEV) cleavage site (His(6)-GB1-lunasin), using pET-25b(+) vector. His(6)-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 H-1 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 mu mol of trolox equivalents/mu mol 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.