School of Chemical Engineering and Advanced Materials, The University of Adelaide, Adelaide, South Australia, Australia.
Division of Research and Innovation, The University of Adelaide, Adelaide, South Australia, Australia.
Biotechnol Bioeng. 2021 Apr;118(4):1707-1720. doi: 10.1002/bit.27687. Epub 2021 Feb 19.
Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus-like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein-DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein-DNA complexes dissociate. Capsomeres thus released show enhanced bind-elute behavior on salt-tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere-DNA complexes yet enables binding to salt-tolerant media. Post purification, assembly experiments indicate that DNA-protein interactions can play a negative role during in vitro assembly, as DNA-protein complexes could not be assembled into virus-like particles, but formed worm-like structures. This study reveals that the control over DNA-protein interaction is a critical consideration during downstream process development for viral vaccines.
在细菌系统中表达病毒衣壳并随后在体外组装成病毒样颗粒是未来负担得起的疫苗的一种可行途径。然而，由于病毒衣壳与色谱介质的结合能力较差，因此纯化具有挑战性。在这项研究中，比较了未分级细菌裂解物中衣壳的行为与纯化衣壳的行为，无论是否添加了微生物 DNA，以更好地了解生物工艺行为不佳的原因。我们表明，衣壳与 DNA 之间的相互作用会形成聚集体或复合物，尤其是在富含污染 DNA 的细菌裂解物中。这些复合物的形成阻止了靶蛋白衣壳进入色谱介质的孔。我们发现可以通过控制缓冲液的离子强度来调节蛋白质-DNA 相互作用，并且在升高的离子强度下，蛋白质-DNA 复合物会解离。因此，释放的衣壳在耐盐色谱介质上表现出增强的结合-洗脱行为。因此，可以有效地去除 DNA。我们相信，这是首次报道使用优化的盐浓度来解离衣壳-DNA 复合物，同时又能与耐盐介质结合。纯化后，组装实验表明，DNA-蛋白质相互作用在体外组装过程中可能会产生负面影响，因为 DNA-蛋白质复合物不能组装成病毒样颗粒，而是形成了蠕虫状结构。这项研究表明，在病毒疫苗的下游工艺开发过程中，控制 DNA-蛋白质相互作用是一个关键考虑因素。
