Stabilizing Disulfide Bridges
Elsewhere at the PEGS event, Alan Dombkowski, Ph.D., assistant professor of clinical pharmacology and toxicology at Wayne State University School of Medicine, discussed techniques for enhancing the thermal stability of proteins through disulfide bond engineering.
He has developed a software package, Software By Design (DbD), that facilitates the rational design of disulfide bonds in proteins.
Nature uses disulfide bonds to stabilize proteins, particularly small secreted proteins that lack a stabilizing hydrophobic core. DbD locates amino acids that are candidates for site-directed mutagenesis that transforms these locations to cysteines, which are then primed for disulfide bond formation. Both intramolecular and intermolecular disulfides are possible, but the latter are more difficult to achieve.
Dr. Dombkowski noted a recent publication in which investigators enhanced the thermal stability of lipase B, which already has a disulfide, by introducing a second sulfur-sulfur linkage. Higher thermal stability provided a more robust process, in this case, for manufacturing biodiesel.
“In most industrial processes, the higher temperatures they can run the reactions at, the better,” Dr. Dombkowski observed. A recent patent application, by DbD licensee Novo Nordisk, describes introduction of a disulfide linkage in growth hormone to make the molecule more resistant to proteolytic degradation. In some cases, disulfide formation improves activity as well, but this is where care must be taken. In a third example Dr. Dombkowski relays, an antibody’s thermal properties were significantly enhanced but activity fell. “The potential effects on activity are real,” he said.
DbD begins with a protein’s structure, and suggest locations where amino acid switches to cysteine are likely to produce a disulfide bridge successfully. The locations must be relatively close in space, but the residues must also possess the correct angle and orientation.
DbD was developed by examining naturally occurring disulfides and characterizing their atomic coordinates, orientations, and geometric requirements, and extrapolating from there to the putative target protein protein. “The software is quite good at modeling these systems, and predicting if the disulfide bond will form,” Dr. Dombroski remarked.
Meantime, Curtis Knox, marketing director at Lucigen, discussed what could be a game-changer for E. coli as an expression system. Available since May, his firm’s CleanColi™ competent cell E. coli strain uses genetically modified lipopolysaccharide (LPS) that does not cause an endotoxic response in humans. Endotoxin removal has been one barrier to employing E. coli as protein expression systems, and using E. coli-derived proteins in cell-based assays.