T4 head-decorating proteins Hoc and Soc do not cause any changes in the Major Capsid Protein arrangement, and their presence is not essential for phage viability. These two proteins have been successfully applied in phage display of foreign proteins and peptides on T4 capsid, mostly as novel vaccine platforms but also for phage purification. Gphoc and gpsoc have also been shown to influence T4 phage susceptibility to cellular uptake. We developed procedures that allow us to obtain highly purified native phage proteins suitable for immunological assays both in vivo and in vitro. Furthermore, we analysed the secondary structure of these proteins by circular dichroism spectroscopy. We also determined the proteins’ stability in increasing concentrations of guanidine chloride. In this work we optimized expression and purification of four bacteriophage T4 head proteins in E. coli, and obtained their immunological purity grade preparations. As was known before, the major head protein gp23 requires assistance of the phage-encoded chaperonin gp31 that is a functional analogue of bacterial GroES protein able to functionally replace it in interaction with the cellular chaperonin GroEL. However, in our experiments this protein was also GW-572016 produced in native conformation using low temperature expression in presence of Cpn10 and Cpn60 chaperones derived from Oleispira antarctica showing 74 and 54% amino acid identity with groEL and groES of E. coli. Recently, the ability of groEL-groES complex to interact with gp23 was reported. Low temperature expression without chaperones yielded an insoluble product, which leads to the suggestion that Cpn10–Cpn60 complex may possess some feature of GroEL-gp31 complex or groEL–groES complex allowing it to assist gp23 folding in T4 infected cells. However, from the practical point of view the best way to produce native gp23 is its co-expression with gp31 protein naturally assisting its folding. Unexpectedly, effective production of gpsoc, the smallest protein in the T4 head, was difficult due to its poor yield when N-terminal fusion of the affinity tag was used. It is possible that some specific sequences present at the gpsoc C-terminus directs this protein to degradation by bacterial proteases. Therefore the soc gene was re-cloned into the expression vector that allowed its expression with a C-terminal affinity tag, resulting in good yield of the protein, but it was almost insoluble. Enhanced expression of chaperone TF improved the solubility to a sufficient level. This result raises a question on the folding of this protein in natural T4 infection. The optimised purification procedure allows for production of nearly homogeneous preparations of all four proteins with very low LPS activity. These proteins have proven native structure and are sufficiently stable, which makes them applicable for immunological studies in vitro and in vivo. Non-essential decorating gphoc represents a typical beta-type secondary structure. This structure has been investigated by in a T4-like bacteriophage, RB49, whose gphoc has only 22% sequence identity with T4.