Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

Abstract

Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.

The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein

“…The nucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI) was from Life Technologies. Lunasin (> 99% purity) was purified from defatted soybean flour (white flake) as previously reported by Kentucky BioProcessing [18]. …”

“…In addition, the majority of the studies were performed using different forms of lunasin including a synthetic lunasin peptide, lunasin enriched soy flour, lunasin-like peptides or a mixture of peptides, rather than a highly purified lunasin isolated from a natural source. The aim of this study was to evaluate lunasin's effect on the proliferation of NSCLC both in vitro and in vivo utilizing highly purified lunasin (>99% purity) isolated from soybean white flake [18]. Our results show for the first time that the in vitro effects of lunasin on NSCLC is significantly higher in anchorage-independent assays, and correlates significantly with its in vivo effects in a NSCLC mouse xenograft model.…”

“…Lunasin, a 43-44 amino acid peptide derived from soybean, consists of nine consecutive aspartic acid residues at the C-terminus, a RGD cell adhesion motif and a helical region exhibiting structural homology to conserved sequences of chromatin binding proteins [18-20]. Although lunasin has been identified in a number of other plants, including barley, wheat, black nightshade ( Solanumnigrum L .…”

GPETAFLR: A new anti-inflammatory peptide from Lupinus angustifolius L. protein hydrolysate

“…MW2 was used for further purification of anti-inflammatory peptides by reversed-phase chromatography (Seber et al, 2012 respectively. Fractions F1-F5 corresponded to unbound peptides and constituted almost 51% of the total mass.…”

1997-2012: Fifteen Years of Research on Peptide Lunasin

“…However, recently, Cavazos and co-workers (2012) have developed an improved method to isolate and purify lunasin from defatted soy flour, resulting in at least 95% purity [23]. Simultaneously, a large-scale method to generate highly purified lunasin from defatted soy flour has been developed by Seber and co-workers (2012) [78]. This method is based on the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography, obtaining preparations of > 99% purity with a yield of 442 mg/kg of defatted soy flour.…”