Effect of recombinant protein expression on baculovirus budded virus structure and infectivity

杆状病毒表达载体(BEVS)是目前应用最广泛、最成功的重组蛋白表达系统之一。通常，在其他系统失败的情况下，他们会成功地制造出困难的目标。它们越来越多地被用于生产重组蛋白亚单位疫苗，例如流感、新冠肺炎和几种动物疾病疫苗。杆状病毒在昆虫细胞中的自然复制周期包括基因表达的早期、晚期和非常晚期。值得注意的是，后期会产生具有感染性的病毒颗粒，这些颗粒从质膜发芽，是病毒在细胞培养中传播的关键。重组蛋白主要是在很晚的阶段产生的，然而，一些蛋白质的合成是在后期开始的，如果靶标是细胞毒性的，这可能会影响芽生病毒(BV)的产量。这可能会将病毒的传染性滴度降低多达10倍。虽然这在监测重组蛋白的小规模测试中通常不是问题，但它可能会在随后的放大工作中产生严重后果，即大量培养的昆虫细胞使用高颗粒：细胞比感染BEVS。另一个问题是生产高滴度的BV股票，这些股票在储存后可能不稳定。这段时间不是保持高滴度数月，而是减少到在冷室中储存几周。病毒低滴度和不稳定性的原因(S)尚不清楚，除了与重组蛋白生产有关外。我们假设BV的结构完整性受到重组蛋白的影响。然而，由于获得最先进设施的机会有限，BV结构数据非常稀缺，导致对这些表达载体缺乏深入了解，并影响BEVS的进一步发展。在STFC、Ral和ISIS拥有世界领先的结构表征设施，我们可以检查和调查感染滴度或储存稳定性受损的重组BV的结构完整性。这项结构研究的成功完成将使我们能够制定重组蛋白生产的智能设计策略，以减轻它们对BV结构和传染性的影响。这将允许目前在可行性阶段失败的项目进一步扩大规模，并为许多不同的学术和商业用途生产蛋白质。

Baculovirus expression vectors (BEVS) have become one of the most versatile and successful systems for the production of recombinant proteins. Frequently, they succeed in producing difficult targets where other systems have failed. Increasingly, they are employed to produce recombinant protein subunit vaccines, e.g. influenza, Covid-19 and several for animal diseases. They are also very useful for producing large, complex transmembrane proteins for drug discovery.The natural replication cycle of baculoviruses in insect cells involves early, late and very late phases of gene expression. Notably, the late phase sees the production of infectious virus particles that bud from the plasma membrane and are essential for virus dissemination in cell culture. Recombinant proteins are largely produced in the very late phase, however, some protein synthesis initiates in the late phase and if the target is cytotoxic this can have an impact on budded virus (BV) yield. This may reduce the infectious titre of virus by up to 10-fold. While this is not usually a problem in small scale tests to monitor recombinant proteins, it can have serious consequences in subsequent scale up work where large cultures of insect cells are infected with BEVS using high particle:cell ratios. A further issue is the production of BV stocks with high titres that may not be stable upon storage. Rather than maintaining a high titre for months, this period reduces to a few weeks of storage in a cold room. The cause(s) of the virus low titre and instability are unknown, apart from the link with recombinant protein production. We hypothesise that the structural integrity of BV is affected by the recombinant protein. However, BV structural data is very scarce due to limited access to state-of-the-art facilities, leading to a lack of in-depth understanding of these expression vectors and impacting on further BEVS development. With world-leading structural-characterisation facilities at STFC RAL and ISIS we can examine and investigate the structural integrity of recombinant BV with compromised infectious titre or stability on storage. The successful completion of this structural study will allow us to formulate intelligent design strategies for recombinant protein production that mitigate their effect on BV structure and infectivity. Thiswill allow projects that currently fail at the feasibilioty stage to progress to scale up and protein production for many different academic and commercial uses.