Therapeutic Protease Engineering
Protease activity is not always a problem to be avoided. Human proteases themselves can be exploited as biologics. The major problems with many applications of so-called therapeutic proteases, however, are that they are not active enough, not specific enough, or are too rapidly inactivated by the highly active serine protease inhibitors, SERPINs, naturally present in human serum.
Wayne M. Coco, vp of protein engineering at Direvo Biotech (www.direvo.com), presented a strategy that uses combinatorial libraries and confocal fluorimetry screening to engineer a nonspecific human protease, trypsin, for high specificity against anti-TNFalpha, a cytokine involved in systemic inflammation. The Direvo scientists accomplished this goal while creating a trypsin variant with an 80-fold increase in specific activity and with several thousand-fold less inhibition in 100% human serum (IC50), according to Coco.
“Our unique combination of library strategies with confocal screening in 100% human serum represents a general platform for the comprehensive optimization of therapeutic proteases,” said Coco. “We can do highly miniaturized, high-throughput screens in formats that are predictive of ultimate application.”
Combinatorial libraries for variant testing included whole gene random mutagenesis and libraries focused on either individual residues or protein domains. “From the improvements that we get in those initial libraries, we recombine the best mutations with proprietary DNA-shuffling techniques,” said Coco.
The Direvo screening technique uses confocal laser technology developed by Evotec (www.evotec.com) and exclusively owned by Direvo for protein engineering. The confocal laser not only detects changes in fluorophore-labeled protein populations, but changes at the individual molecule level, thus contributing to miniaturization, throughput, and unique assay formats.