Granzyme B, a 247 amino acid polypeptide, contains a leader sequence, which is cleaved by a signal peptidase and a two amino acid prodomain, which is cleaved by the lysosomal cysteine protease DPPI. The recombinant granzyme B is expressed in Pichia pastoris as the mature form and appears on SDS-PAGE as a triplet (~34, 32, and 30 kDa) due to three different glycosylations.

Cytotoxic granules secreted by natural killer (NK) cells and cytotoxic T Lymphocytes cells are part of the mechanism used for protecting the organism from virus infected and tumor cells. Granzyme B, a serine protease, is the most prominent granzyme in a family of 11 found in the cytotoxic granules. The granzymes enter the target cell with the assistance of perforin, a critical molecule of the cytotoxic granules. In the target cell, the granzymes act on specific substrates involved with the cell death via apoptosis.
Granzyme B is a neutral serine protease that cleaves aspartic acid residues, inducing cell death by various pathways. It can cleave and activate most of the caspases in vitro and in vivo resulting in a massive amplification of the caspase dependent apoptotic pathway. In addition, granzyme B cleaves directly downstream caspase substrates as PARP DNA-PK2 and DFF45/ICAD leading to cell death. This pathway bypasses inhibition of apoptosis by viral caspase inhibitors found in virus infected cells. It has been shown that granzyme B is capable of inducing cytochrome C release from the mitochondria in a caspase independent way.

Pichia expressed recombinant Granzyme B has the advantage over native Granzyme B as the latter can be purified only in limited amounts, and over E. coli and vaccinia virus expressed enzymes that failed to generate soluble active enzyme.

One unit will hydrolyze 1.0 nmole of Boc-Ala-Ala-Asp-sBzl per min at 30 °C.

Solution in 50 mM HEPES, pH 7.5 , containing 150 mM NaCl.

Recombinant Granzyme B expressed in Pichia pastoris is a soluble secreted form of the active enzyme with the same specificities as native Granzyme B. This recombinant, active, mature Granzyme B can cleave caspase 3 both in vitro and in vivo to its signature p20/p10 forms.