Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of ) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in markedly improved detection of this protein. We therefore hypothesized that transcripts containing may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of that had been codon-optimized for , yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in Interestingly, expression of non-optimized produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in and bacteria, produced increased green fluorescence compared to untagged (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

中文翻译：

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鉴定可改善土拉弗朗西斯菌和其他革兰氏阴性菌荧光蛋白生物合成的 N 末端标签 (580N)

荧光蛋白广泛用于微生物发病机制和宿主-病原体相互作用的研究。在这里，我们发现 FTL_0580（一种假设的蛋白质）的 36 个 N 端氨基酸与荧光蛋白的连接增加了表达这些重组融合体的细菌的荧光发射。该N-末端肽将被称为580N。蛋白质印迹显示 580N 与翠绿荧光蛋白 (EmGFP) 的连接显着改善了该蛋白的检测。因此，我们假设包含该前导肽的转录物可能比那些缺乏该前导肽的编码序列的转录物更有效地翻译。作为支持，其表达已针对 进行了密码子优化，与未优化的对应物相比，产生了显着增强的荧光。此外，与之前已被证明可以抑制核糖体停滞的小N端肽（丝氨酸-赖氨酸-异亮氨酸-赖氨酸）的编码序列融合，在表达时产生强烈的荧光。这些发现支持了580N增强翻译效率的解释。有趣的是，未优化的表达产生比任何其他构建体更大的荧光强度。结构预测表明 RNA 二级结构也可能影响翻译效率。当在细菌中表达时，与未标记的细菌相比，产生增加的绿色荧光（两个等位基因都没有针对这些细菌进行密码子优化）。总之，将 580N 前导肽的编码序列与重组基因融合可能作为密码子优化的经济替代方案，以增强细菌中的蛋白质表达。