Huang Kelly, Lawlor Heather, Tang Roderick, MacGill Randall S, Ulbrandt Nancy D, Wu Herren
MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
Virus Genes. 2010 Apr;40(2):212-21. doi: 10.1007/s11262-010-0449-8. Epub 2010 Jan 29.
Studies of the fusion activity of respiratory syncytial virus (RSV) F protein are significantly hindered by low recombinant expression levels. While infection produces F protein levels detectable by western blot, recombinant expression produces undetectable to low levels of F protein. Identifying the obstacles that hinder recombinant F protein expression may lead to improved expression and facilitate the study of F protein function. We hypothesized that nuclear localization and/or inefficient RNA polymerase II-mediated transcription contribute to poor recombinant F protein expression. This study shows a combination of stalled nuclear export, premature polyadenylation, and low mRNA abundance all contribute to low recombinant F protein expression levels. In addition, this study provides an expression optimization strategy that results in greater F protein expression levels than observed by codon-optimization of the F protein gene, which will be useful for future studies of F protein function.
呼吸道合胞病毒（RSV）F蛋白融合活性的研究因重组表达水平低而受到显著阻碍。虽然感染产生的F蛋白水平可通过蛋白质免疫印迹法检测到，但重组表达产生的F蛋白水平却检测不到或很低。识别阻碍重组F蛋白表达的障碍可能会提高表达水平并促进对F蛋白功能的研究。我们推测核定位和/或RNA聚合酶II介导的转录效率低下导致重组F蛋白表达不佳。本研究表明，停滞的核输出、过早的聚腺苷酸化和低mRNA丰度共同导致重组F蛋白表达水平低下。此外，本研究提供了一种表达优化策略，该策略所产生的F蛋白表达水平高于对F蛋白基因进行密码子优化所观察到的水平，这将有助于未来对F蛋白功能的研究。
