PRODUCTION OF FULLY ACTIVE RECOMBINANT MURINE GRANZYME-B IN YEAST

Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymp hocytes; this enzyme is critically involved in delivering the rapid ap optotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for i ts activation. In this report, we have successfully produced fully act ive, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusin g GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX 2 signal peptidase to release the processed enzyme into the supernatan t of yeast cultures, We expressed the proenzyme form of GzmB as well a nd determined that pro-GzmB is efficiently converted to its active for m by the cysteine proteinase dipeptidyl peptidase I, The fully process ed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxyca rbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a k(cat) of 17 s(-1) and catalytic efficiency k(cat)/K-m of 181, 237 M-1 s(-1); the recombinant enzyme is therefore at least twice as a ctive as purified native GzmB, In addition, the recombinant enzyme hyd rolyzes Boc-Ala-Ala-Met thiobenzyl ester with a K-cat of 3.2 s(-1) and a catalytic efficiency k(cat)/K-m of 65,306 M-1 s(-1). Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p 20/p10 forms, Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK as well as Zn2+ (a known inhibitor of caspase-3). Struc tural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.