Recombinant production of the therapeutic peptide lunasin

Kyle Stuart, James, Mcpherson

Description

Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an Arg-Gly-Asp cell adhesion motif and eight aspartic acid residues. In vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting the Clostridium thermocellum CipB cellulose binding domain (CBD) as a fusion partner protein. Results We used a pET28 vector to express a CBD-lunasin fusion with a hexahistidine tag and Tobacco Etch Virus protease site, to allow protease-mediated release of native lunasin. Autoinduction in E. coli BL21 (DE3) Star cells achieved expression of 3.35 g/L of CBD-lunasin fusion protein. The final yield of lunasin was 210 mg/L corresponding to 32% of the theoretical yield. Purification by cellulose binding and nickel affinity chromatography were tested with the latter proving more satisfactory. The effects of CBD-lunasin expression on growth and morphology of the E. coli cells were examined by light and electron microscopy revealing an altered morphology in a proportion of cells. Cell division appeared to be inhibited in these cells resulting in elongated, non-septated cells. Conclusions The use of CBD as a fusion partner gave high protein yields by autoinduction, with lunasin release by TEV protease cleavage. With some optimisation this approach could provide a potentially valuable route for production of this therapeutic peptide. Over-expression in the host cells manifest as a cell division defect in a population of the cells, presumably mimicking some aspect of the chemopreventive function observed in mammalian cells.

Sujets

• E. coli

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Kyleet al.Microbial Cell Factories2012,11:28 http://www.microbialcellfactories.com/content/11/1/28

R E S E A R C HOpen Access Recombinant production of the therapeutic peptide lunasin * Stuart Kyle, Kier AR James and Michael J McPherson

Abstract Background:Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an ArgGlyAsp cell adhesion motif and eight aspartic acid residues.In vitrostudies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting theClostridium thermocellumCipB cellulose binding domain (CBD) as a fusion partner protein. Results:We used a pET28 vector to express a CBDlunasin fusion with a hexahistidine tag and Tobacco Etch Virus protease site, to allow proteasemediated release of native lunasin. Autoinduction inE. coliBL21 (DE3) Star cells achieved expression of 3.35 g/L of CBDlunasin fusion protein. The final yield of lunasin was 210 mg/L corresponding to 32% of the theoretical yield. Purification by cellulose binding and nickel affinity chromatography were tested with the latter proving more satisfactory. The effects of CBDlunasin expression on growth and morphology of theE. colicells were examined by light and electron microscopy revealing an altered morphology in a proportion of cells. Cell division appeared to be inhibited in these cells resulting in elongated, nonseptated cells. Conclusions:The use of CBD as a fusion partner gave high protein yields by autoinduction, with lunasin release by TEV protease cleavage. With some optimisation this approach could provide a potentially valuable route for production of this therapeutic peptide. Overexpression in the host cells manifest as a cell division defect in a population of the cells, presumably mimicking some aspect of the chemopreventive function observed in mammalian cells. Keywords:Lunasin, Autoinduction,E. coli, Cellulose binding domain, Chemopreventive peptide

Background Lunasin is a 43 amino acid chemopreventive peptide initially identified in soybean and more recently in bar ley, wheat, andSolanum nigrum[1]. It is a small subunit peptide derived from the larger cotyledonspecific 2S albumin (Gm2S1) complex and comprises three distinct regions. An Nterminal helical region that shares homol ogy with the conserved region of the chromatin binding proteins, an ArgGlyAsp (RGD) cell adhesion motif and a Cterminal sequence of eight aspartic acid (D) residues [2]. Various studies have shown the ability of lunasin to suppress both chemical and oncogene driven transfor mation of mammalian cells. The chemopreventive

* Correspondence: [e-mail] Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

properties have also been illustrated in mouse skin can cer models where lunasin has been shown to suppress transformation and subsequent carcinogenesis [3]. The proposed biological activity of the regions of lunasin is shown in Figure 1. There are two broad strategies for the production of therapeutic peptides and proteins: chemical synthesis and recombinant production by transgenic organisms ranging from bacteria and fungi to plants and animals. Chemical synthesis is rapid and effective for the produc tion of custommade peptides in relatively small quanti ties but can be costly and problematic during process scaleup and as amino acid sequence length increases with sequences over 35 amino acids not generally con sidered to be economically feasible [5]. In addition, the process employs chemicals that present potential envir onmental hazards. The use of microbial‘biofactories’for

© 2012 Kyle et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.