Proteome profiling and vector yield optimization in a recombinant adeno‐associated virus‐producing yeast model,MicrobiologyOpen

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Proteome profiling and vector yield optimization in a recombinant adeno‐associated virus‐producing yeast model
MicrobiologyOpen ( IF 3.9 ) Pub Date : 2020-11-09 , DOI: 10.1002/mbo3.1136
Juan Jose Aponte-Ubillus _{1,

2} , Daniel Barajas ₁ , Harry Sterling ₁ , Ali Aghajanirefah ₁ , Cameron Bardliving ₂ , Joseph Peltier ₁ , Parviz Shamlou ₂ , Mimi Roy ₁ , Daniel Gold ₁

Affiliation

Recent studies on recombinant adeno‐associated viral (rAAV) vector production demonstrated the generation of infectious viral particles in Saccharomyces cerevisiae. Proof‐of‐concept results showed low vector yields that correlated with low AAV DNA encapsidation rates. In an attempt to understand the host cell response to rAAV production, we profiled proteomic changes throughout the fermentation process by mass spectrometry. By comparing an rAAV‐producing yeast strain with a respective non‐producer control, we identified a subset of yeast host proteins with significantly different expression patterns during the rAAV induction period. Gene ontology enrichment and network interaction analyses identified changes in expression patterns associated mainly with protein folding, as well as amino acid metabolism, gluconeogenesis, and stress response. Specific fold change patterns of heat shock proteins and other stress protein markers suggested the occurrence of a cytosolic unfolded protein response during rAAV protein expression. Also, a correlative increase in proteins involved in response to oxidative stress suggested cellular activities to ameliorate the effects of reactive oxygen species or other oxidants. We tested the functional relevance of the identified host proteins by overexpressing selected protein leads using low‐ and high‐copy number plasmids. Increased vector yields up to threefold were observed in clones where proteins SSA1, SSE1, SSE2, CCP1, GTT1, and RVB2 were overexpressed. Recombinant expression of SSA1 and YDJ insect homologues (HSP40 and HSC70, respectively) in Sf9 cells led to a volumetric vector yield increase of 50% relative to control, which validated the importance of chaperone proteins in rAAV‐producing systems. Overall, these results highlight the utility of proteomic‐based tools for the understanding and optimization of rAAV‐producing recombinant strains.

中文翻译：

重组腺相关病毒生产酵母模型中的蛋白质组分析和载体产量优化

最近对重组腺相关病毒 (rAAV) 载体生产的研究表明，酿酒酵母中会产生感染性病毒颗粒. 概念验证结果显示低载体产量与低 AAV DNA 衣壳化率相关。为了了解宿主细胞对 rAAV 产生的反应，我们通过质谱分析了整个发酵过程中的蛋白质组学变化。通过将产生 rAAV 的酵母菌株与各自的非生产者对照进行比较，我们确定了在 rAAV 诱导期间具有显着不同表达模式的酵母宿主蛋白子集。基因本体富集和网络相互作用分析确定了主要与蛋白质折叠、氨基酸代谢、糖异生和应激反应相关的表达模式的变化。热休克蛋白和其他应激蛋白标记物的特定倍数变化模式表明在 rAAV 蛋白表达过程中发生了胞质未折叠蛋白反应。此外，与氧化应激反应有关的蛋白质的相关增加表明细胞活动可以改善活性氧或其他氧化剂的影响。我们通过使用低拷贝数和高拷贝数质粒过表达选定的蛋白质先导来测试已识别宿主蛋白质的功能相关性。在蛋白 SSA1、SSE1、SSE2、CCP1、GTT1 和 RVB2 过表达的克隆中观察到载体产量增加了三倍。Sf9 细胞中 SSA1 和 YDJ 昆虫同源物（分别为 HSP40 和 HSC70）的重组表达导致相对于对照的体积载体产量增加 50%，这证实了伴侣蛋白在 rAAV 产生系统中的重要性。总的来说，这些结果突出了基于蛋白质组学的工具在理解和优化产生 rAAV 的重组菌株方面的实用性。

更新日期：2020-12-23

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