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

Abstract

SummaryHuman transforming growth factor-b3 (TGFb3) 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 TGFb3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFb3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFb3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFb3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFb3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFb3 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 TGFb3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFb3 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 TGFb3. 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 TGFb3.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…The first codons immediately downstream from the AUG start codon have already been identified as an important regulator of translation efficiency in chloroplasts (Kuroda and Maliga 2001). A recent study showed codon optimization of the TGFb3 leading to a 75-fold increase in recombinant protein accumulation relative to the native human coding sequence (Gisby et al 2011).…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Franklin et al (2002) reported a lack of variation in transcript abundance for GFP expression in Chlamydomonas reinhardtii chloroplasts despite an 80-fold increase in GFP protein accumulation of the codonoptimized sequence. Even though there was a 3-fold increase in mRNA levels of codon-optimized TGF-b3 when compared with the native sequence (Gisby et al, 2011), the greater part of the 75-fold increase in synthetic TGF-b sequence was attributed to enhanced translation. A recent study also showed that the compatibility of the 5ʹ UTR and its coding sequence increased the efficient translation of codon-optimized sequences rather than mRNA abundance (Nakamura et al, 2016).…”

“…Northern blots indicated that the increase of codon-optimized CNTB-FVIII HC and CNTB-VP1 accumulation is at the translational level rather than RNA transcript accumulation. Several previous studies on the expression of foreign genes have shown a lack of variation or modest increases in transcript abundance but significant variation in translation efficiency (Franklin et al, 2002;Gisby et al, 2011;Nakamura et al, 2016). Franklin et al (2002) reported a lack of variation in transcript abundance for GFP expression in Chlamydomonas reinhardtii chloroplasts despite an 80-fold increase in GFP protein accumulation of the codonoptimized sequence.…”

“…The CNTB-fused native sequence of human proinsulin (approximately 22 kD) was expressed up to 72% of total leaf protein (Ruhlman et al, 2010), and the expression of ZZTEV-IGF-1 (Staphylococcus aureus Z domains and TEV cleavage site fused to native human insulin-like growth factor1 gene; approximately 26 kD) was up to 32.4% of TSP (Daniell et al, 2009). However, human TGF-b3 (13 kD, 56% GC) was expressed in up to 12% of leaf protein only after codon optimization (Gisby et al, 2011). Also, the expression of GFP (approximately 26 kD) increased approximately 80-fold after codon optimization (Franklin et al, 2002).…”

“…In vitro assays of inserted genes with several synonymous codons show that translation efficiency does not always correlate with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007), but they have been used in several codon optimization studies (Lutz et al, 2001;Ye et al, 2001;Franklin et al, 2002;Lenzi et al, 2008;Jabeen at al., 2010;Madesis et al, 2010;Gisby et al, 2011;Wang et al, 2015b;Boehm et al, 2016;Nakamura et al, 2016). While some studies achieved significant increases in expression (75-to 80-fold) after codon optimization (Franklin et al, 2002;Gisby et al, 2011), other studies observed negligible enhancement (Ye et al, 2001;Lenzi et al, 2008;Daniell et al, 2009;Wang et al, 2015b;Nakamura et al, 2016).…”