RESULTS: Amino acid sequence analysis indicated 2,611 SPs from the TGF-β superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12.
CONCLUSIONS: These results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.
结果:氨基酸序列分析表明,当与BMP2连接时,来自TGF-β超家族的2,611个SP具有功能。mRNA结构预测表明翻译起始位点的结构可能高度可变。具有最易接近的翻译起始位点的五个序列,密码子优化的BMP2SP变体和完善的hIL2SP序列被用于体外测试。前五个候选物显示HEK293T细胞中BMP2分泌的非显著改善。与C2C12细胞中的天然序列相比,全部显示分泌减少,有几个显示出大且显著的下降。在C2C12s中,没有一个测试序列能够增加碱性磷酸酶活性高于背景。密码子优化的对照序列和hIL2SP在HEK293T中显示出合理的活性,但在C2C12中显示出非常差的活性。
结论:这些结果支持使用基于肽序列的计算机工具,在合成生物学背景下对信号肽活性进行基本预测。然而,mRNA结构预测需要改进才能为该应用产生可靠的预测。C2C12中密码子优化的BMP2SP变体的低活性强调了密码子选择的重要性,mRNA结构,和SP活动的细胞环境。