A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule

Plant Biotechnol J. 2011 Jun;9(5):618-28. doi: 10.1111/j.1467-7652.2011.00619.x. Epub 2011 Apr 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chloroplasts / genetics*
  • Chloroplasts / metabolism*
  • Gene Expression Regulation, Plant
  • Genes, Synthetic*
  • Humans
  • Molecular Sequence Data
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Protein Conformation
  • Protein Engineering / methods
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Tobacco / genetics
  • Tobacco / metabolism
  • Transformation, Genetic
  • Transforming Growth Factor beta3 / biosynthesis*
  • Transforming Growth Factor beta3 / chemistry*
  • Transforming Growth Factor beta3 / genetics
  • Transforming Growth Factor beta3 / isolation & purification

Substances

  • Recombinant Proteins
  • TGFB3 protein, human
  • Transforming Growth Factor beta3