Many mammalian proteins can be expressed at high levels in the prokaryotic host E. coli, but frequently accumulate in a misfolded and biologically inactive form in inclusion bodies. Protein solubility can either be enhanced by modifying the production process, or by protein engineering. Most SCH772984 efforts have been directed to chemical denaturation of purified inclusion body proteins followed by refolding procedures.However,this strategy needs tobe adapted toa particular protein species, and the yields of soluble protein can vary considerably. Attempts to prevent inclusion body formation and directly increase the solubility of recombinant proteins include expression of the protein of interest fused to a heterologous protein domain as a solubilizing agent. Protein domains employed in this manner comprise glutathione-S-transferase from Schistosoma japonicum, thioredoxin, ubiquitin, protein A, DsbA, domain 1 of the translation initiation factor IF2, and the maltosebinding protein of E. coli. MBP is part of a large class of proteins that aid in the uptake of small molecules. While it naturally resides in the periplasm, MBP can also be expressed at high yields in the cytoplasm. For different proteins increased solubility, enhanced stability and markedly improved yields have been reported after fusion to MBP. This has been explained by the ability of MBP to act as a chaperone in the context of a fusion protein, and promote the proper folding of the fusion partner. Here we investigated the potential of MBP to improve as part of a fusion protein the expression of human granzyme B as a secreted recombinant protein in the methylotrophic yeast Pichia pastoris. The serine protease GrB is normally released by cytotoxic lymphocytes. It enters target cells together with the pore-forming protein perforin and induces apoptosis by activating caspasedependent and caspase-independent programmed cell death pathways. The availability of GrB in recombinant form is an important prerequisite for functional analysis, and is essential for application of GrB as a therapeutic effector protein. P. pastoris has previously been employed to generate recombinant GrB from different mammalian species. This yeast represents a widely established eukaryotic expression system for proteins that are secreted to the extracellular space. Fusion of the yeast mating type a-factor signal peptide to the protein of interest thereby directs it to the secretory pathway, where it becomes glycosylated before release into the culture supernatant. GrB derivatives have also been expressed as GST fusion proteins in E. coli. Thereby proteolytic processing of such fusion proteins at a cleavage site introduced between GrB and GST domains was required.