To　investigate　the　expression　and　purification　of　an　unstable　heterologous　protein　in　Pichia　pastoris,　the　cDNA　of　H5-lysozyme,　a　hen　egg　lysozyme　mutant　with　a　hydrophobic　pentapeptide　(Phe-Phe-Val-Ala-Pro)　fused　to　the　carboxyl　terminus,　was　integrated　into　the　genome　of　P.　pastoris.　It　was　found　that　medium　composition,　induction　time,　and　fermenter　type　were　important　factors　for　the　expression　of　H5-lysozyme.　Substantially　active　H5-lysozyme　was　secreted　by　induction　with　methanol　when　the　prepro-sequence　of　alpha-factor　was　used　as　secretion　signal　sequence.　The　amount　secreted　was　422-fold　greater　than　that　observed　with　Saccharomyces　cerevisiae.　Recombinant　H5-lysozyme　was　recovered　and　purified　by　cation-exchange　chromatography　directly　from　fermentation　broth.　The　mutant　lysozyme　showed　bactericidal　activity　against　Gram-positive　as　well　as　Gram-negative　bacteria.　(C)　2002　Elsevier　Science　(USA).　All　rights　reserved.