title: A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.
abstract: Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFβ3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, UK.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Gisby
authors.forename: Philip
authors.initials: P
authors.lastname: Mellors
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Madesis
authors.forename: Marianne
authors.initials: M
authors.lastname: Ellin
authors.forename: Hugh
authors.initials: H
authors.lastname: Laverty
authors.forename: Sharon
authors.initials: S
authors.lastname: O'Kane
authors.forename: Mark W J
authors.initials: MW
authors.lastname: Ferguson
authors.forename: Anil
authors.initials: A
authors.lastname: Day
mesh_terms: D001483:Base Sequence; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D053782:Transforming Growth Factor beta3 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; C509044:TGFB3 protein, human; D053782:Transforming Growth Factor beta3