| Autor: |
Gisby, Martin F., Mellors, Philip, Madesis, Panagiotis, Ellin, Marianne, Laverty, Hugh, O'Kane, Sharon, Ferguson, Mark W J, Day, Anil |
| Jazyk: |
angličtina |
| Rok vydání: |
2011 |
| Předmět: |
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| Zdroj: |
Gisby, M F, Mellors, P, Madesis, P, Ellin, M, Laverty, H, O'Kane, S, Ferguson, M W J & Day, A 2011, ' A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule ', Plant Biotechnology Journal, vol. 9, no. 5, pp. 618-628 . https://doi.org/10.1111/j.1467-7652.2011.00619.x |
| DOI: |
10.1111/j.1467-7652.2011.00619.x |
| Popis: |
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. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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