The tail sheath protein of giant bacteriophage φKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion by trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of φKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4. © 2009 Elsevier Inc.