Griswold Karl E, Mahmood Nadir A, Iverson Brent L, Georgiou George
Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
Protein Expr Purif. 2003 Jan;27(1):134-42. doi: 10.1016/s1046-5928(02)00578-8.
Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria. However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E. coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E. coli codons in the fungal sequence. On the other hand, expression in the E. coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E. coli optimized and wild-type constructs. Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E. coli optimized versus wild-type cutinase genes is due to differences in 5(') mRNA secondary structures. In particular, our results indicate that increased stability of 5(') mRNA secondary structures in the E. coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence. These results underscore the idea that potential 5(') mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E. coli.
使重组基因的密码子使用与表达宿主的密码子使用相匹配，是提高细菌中外源蛋白表达水平的常用策略。然而，在开发用于在大肠杆菌中表达茄病镰刀菌角质酶的胞质表达系统时，我们发现，尽管真菌序列中存在几个大肠杆菌稀有密码子，但将密码子改变为大肠杆菌偏好的密码子后，与野生型真菌基因相比，表达水平显著降低。另一方面，使用细菌PhoA前导序列在大肠杆菌周质中表达时，大肠杆菌优化型和野生型构建体均实现了高水平表达。序列交换实验以及预测的mRNA二级结构计算结果支持了以下假设：大肠杆菌优化型与野生型角质酶基因在胞质中的表达差异是由于5′端mRNA二级结构不同所致。特别是，我们的结果表明，在没有phoA前导序列的情况下，大肠杆菌优化型转录本中5′端mRNA二级结构稳定性的增加会阻止有效的翻译起始。这些结果强调，在设计用于在大肠杆菌中高水平表达的合成基因时，应同时考虑潜在的5′端mRNA二级结构和密码子使用情况。
