Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

Lauren E Seber,Steven D Hume,Keith R Davis,Brian W Barnett,Elizabeth J Mcconnell,Jian Cai,Kati Boles,Maxim Antopolsky

doi:10.1371/journal.pone.0035409

Lauren E Seber, Steven D Hume + Show 6 more

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https://doi.org/10.1371/journal.pone.0035409

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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.

Highlights

Lunasin has been described as a 43 amino-acid peptide that is encoded within the soybean GM2S-1 gene and was first identified as a novel peptide found in soybean seed extracts [1]
Initial studies of the biological activity of lunasin found that expression constructs encoding the lunasin peptide sequence resulted in arrested cell division and the formation of nonseptated filaments in E. coli and caused mitotic arrest in mammalian cell lines, apparently by binding to kinetochore regions of the centromere and blocking microtubule attachment [2]
Establishment of extraction conditions Previous reports describing the partial purification of lunasin utilized extraction of soy flour with water and phosphate buffered saline (PBS) [15,30,31]; a systematic analysis of extraction conditions was not described

Summary

Introduction

Lunasin has been described as a 43 amino-acid peptide that is encoded within the soybean GM2S-1 gene and was first identified as a novel peptide found in soybean seed extracts [1]. Initial studies of the biological activity of lunasin found that expression constructs encoding the lunasin peptide sequence resulted in arrested cell division and the formation of nonseptated filaments in E. coli and caused mitotic arrest in mammalian cell lines, apparently by binding to kinetochore regions of the centromere and blocking microtubule attachment [2]. These initial results suggested that lunasin could be useful as a cancer therapeutic provided that lunasin could be delivered to cancer cells. Analysis of different soybean cultivars demonstrated that lunasin content varied significantly, suggesting that it may be possible to breed soybean varieties with higher lunasin content [14,15]

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