Protein V is an adenovirus core protein, having a role in condensing adenovirus DNA and protein VII complex within the capsid. More specifically, it functions as a shell around the DNA and core proteins, establishing a link between core and capsid proteins by acting as a bridge. The literature reveals that protein V has been successfully extracted numerous times from mammalian cells; however, only few investigators have attempted to purify it from bacterial systems. This was due to several factors, including the cationic nature of the protein (pI = 10.34), which makes it extremely hard to purify from E. coli. With the aim to develop a high yield expression and purification protocol, protein V was cloned in a pET vector with a thioredoxin fusion tag, and purified successfully using a HisTrap IMAC column followed by Phenyl HP hydrophobic interaction column purification.