期刊
PLANT BIOTECHNOLOGY JOURNAL
卷 9, 期 5, 页码 618-628出版社
WILEY
DOI: 10.1111/j.1467-7652.2011.00619.x
关键词
plastid transformation; transforming growth factor; codon usage; chloroplast transformation; Nicotiana
资金
- Biotechnology and Biological Sciences Research Council
- BBSRC [BB/E020445/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E020445/1] Funding Source: researchfish
P>Human transforming growth factor-beta 3 (TGF beta 3) 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 TGF beta 3 in chloroplasts and its subsequent purification would provide a sustainable source of TGF beta 3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGF beta 3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGF beta 3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGF beta 3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGF beta 3 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 TGF beta 3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGF beta 3 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 TGF beta 3. 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 TGF beta 3.
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