Adenoviral proteins V and VII (pV and pVII) play important roles in condensing the viral DNA within the capsid protein, hexon. More specifically, pV functions as a shell around the viral DNA and core proteins, establishing a link between core and capsid proteins by acting as a bridge, whereas pVII has been reported to act as a histone-like protein by condensing DNA. The literature reveals that protein V and VII have been extracted from adenovirus, however their expression and purification in bacteria have been extremely challenging. For this reason structural data is not available for these proteins, even though they have been reported some 50 years ago. This was due to several factors, including the cationic nature of the protein (pV: pI = 10.34; pVII: pI = 12.34), which makes it extremely hard to purify them from bacterial expression systems.
With the aim to develop a high yield expression and purification protocol, pV and pVII were cloned in three different vectors to scrutinize the expression and purification level of these proteins. The vectors were a gateway vector with 6xHis-tag, a pET vector with thioredoxin and 6xHis-tag, and a pET vector with GST tag. The gateway vectors were found to express the highest level of proteins in E. coli BL21 (DE3) cells; however, in terms of both expression and purification the thioredoxin vectors were efficient and so preferred. Interestingly, we found that these core proteins have high affinity to nickel bead columns and so can’t be purified efficiently through 6xHis-tag based affinity chromatography in higher concentrations. Furthermore, all of the constructs except thioredoxin pV expressed protein in soluble form, however, the 6xHis-tag of the proteins were hidden and not freely available to bind to Ni2+. To overcome this issue, the proteins were denatured with 6M Gdn-HCl and then refolded before functional assays were performed.