title: Recombinant production of the therapeutic peptide lunasin.
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 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.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: Kyle
authors.forename: Kier A R
authors.initials: KA
authors.lastname: James
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McPherson
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002846:Chromatography, Affinity; D048013:Acetivibrio thermocellus / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C556331:lunasin protein, barley; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease