Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.

中文翻译：

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从叶绿体基质易位到类囊体腔中使得重组表皮生长因子能够在转质体烟草植物中表达。


叶绿体转化对于在植物中生产重组蛋白具有许多潜在的优势。然而，据报道，许多蛋白质的叶绿体表达，例如人表皮生长因子（hEGF），会受到转录后机制的阻碍。 hEGF 降解与基质的氧化还原电位和蛋白质错误折叠有关。为了解决这个问题，我们提出将 hEGF 重定向到类囊体腔中，其中的环境可以改善二硫键的形成，从而稳定蛋白质的功能构象。我们通过添加转运肽 (Str) 生成了将 hEGF 蛋白靶向类囊体腔的转质体烟草植物。按照这种方法，我们可以通过蛋白质印迹检测类囊体腔靶向的 hEGF，而 hEGF 的基质积累仍然检测不到。 Southern印迹分析证实转基因通过同源重组整合到质体中。 Northern印迹分析显示EGF和StrEGF系中的egf转录物水平相似。这些结果表明 hEGF 肽在类囊体腔中的较高稳定性是在 StrEGF 系中观察到的重组蛋白积累增加的主要原因。他们还强调了探索不同亚细胞器目的地以提高质体中特定重组蛋白积累水平的必要性。