Kheirvari Milad, Tumban Ebenezer
Graduate Program in One Health Sciences, School of Veterinary Medicine, Texas Tech University, Amarillo, TX 79106, USA.
Int J Mol Sci. 2025 Sep 6;26(17):8688. doi: 10.3390/ijms26178688.
P23-77 is a thermophilic bacteriophage that infects bacteria. The genome of the virus is enclosed in an icosahedral capsid. This capsid is made of the small major capsid protein (VP16), the large major capsid protein (VP17), and the minor capsid protein (VP11). In addition to these three structural proteins, membrane-associated proteins (VP15, VP19, VP20, VP22, and VP23) have been identified in the virus and may serve as scaffold proteins to help with viral assembly. Previous studies have expressed VP11, VP16, and VP17 in . A mixture of these proteins can lead to the formation of complexes. However, the potential to express membrane-associated proteins has never been explored. Here, we demonstrated, for the first time, the expression and co-expression of some membrane-associated proteins with capsid (coat) proteins, both in the natural host and in . Co-expression of these proteins did not result in the assembly of virus-like particles. We explored further strategies to express and purify some of the proteins for future studies. We observed that the insertion of a purification tag (Strep-II tag, but not a histidine tag) significantly reduced the expression levels of some of the proteins. Six of the eight structural proteins were successfully purified to homogeneity using different approaches. We showed that VP20 and VP22 migrated on SDS PAGE gel at sizes larger than their predicted molecular weights. Predicted 3D structures of the proteins show that most of them are helical in nature with disordered regions. The work presented here will help pave the way for the expression and purification of these proteins. This will help determine their 3D structures and may shed light on the requirements for viral assembly.
P23 - 77是一种感染细菌的嗜热噬菌体。该病毒的基因组被包裹在一个二十面体衣壳中。这个衣壳由小的主要衣壳蛋白（VP16）、大的主要衣壳蛋白（VP17）和次要衣壳蛋白（VP11）组成。除了这三种结构蛋白外，还在病毒中鉴定出了膜相关蛋白（VP15、VP19、VP20、VP22和VP23），它们可能作为支架蛋白来帮助病毒组装。先前的研究已经在……中表达了VP11、VP16和VP17。这些蛋白质的混合物可导致复合物的形成。然而，从未探索过表达膜相关蛋白的潜力。在这里，我们首次证明了一些膜相关蛋白与衣壳（外壳）蛋白在天然宿主和……中的表达及共表达。这些蛋白质的共表达并未导致病毒样颗粒的组装。我们探索了进一步的策略来表达和纯化其中一些蛋白质以供未来研究。我们观察到插入一个纯化标签（链霉亲和素 - II标签，但不是组氨酸标签）显著降低了其中一些蛋白质的表达水平。使用不同方法成功地将八种结构蛋白中的六种纯化至同质。我们发现VP20和VP22在SDS - PAGE凝胶上迁移时的大小比其预测的分子量更大。预测的蛋白质三维结构表明，它们中的大多数本质上是螺旋状的，带有无序区域。本文所呈现的工作将有助于为这些蛋白质的表达和纯化铺平道路。这将有助于确定它们的三维结构，并可能揭示病毒组装的要求。
