Design, Co-Expression, and Evaluation for Assembly of the Structural Proteins from Thermophilic Bacteriophage ΦIN93

Bacteriophage ΦIN93 has an icosahedral-like capsid that is believed to be composed of two putative capsid or coat proteins, namely open reading frame (ORF)13 and ORF14. In addition to the two capsid proteins, there are other proteins that may be associated with the structure of the virus. For example, five other proteins (ORF12, ORF16, ORF17, ORF19, and ORF20) in the virus have been identified as putative membrane-associated proteins. It is believed that membrane-associated proteins associate with coat proteins (serve as scaffolding proteins) to promote viral assembly. While the expression/co-expression of ORF13 and ORF14 have been done to assess if they can assemble to form virus-like particles (VLPs), the expression of any of the membrane-associated proteins and their contribution to assembly have never been attempted. In this study, we successfully co-expressed, for the first time, three membrane-associated proteins (ORF12, ORF16, ORF17) in addition to ORF13 and ORF14 in thermophilic bacteria (Thermus thermophilus, HB27:nar strain) and in mesophilic bacteria (BL21 Star). The expression levels of the proteins were higher in BL21 Star than in Thermus thermophilus, HB27:nar. Some of the expressed proteins (especially ORF17) migrated at sizes that were more than their deduced molecular weight (based on amino acid sequence). Co-expression of these proteins did not lead to the formation of structures that we believe are VLPs. Nevertheless, we believe co-expressing these proteins together from different plasmids is a good approach to assess which of them may be required to form VLPs.