Effects of Shear Rate and Protein Concentration on Amyloidogenesis via Interfacial Shear.

The influence of hydrodynamics on protein fibrillization kinetics is relevant to biophysics, biochemical reactors, medicine, and disease. This investigation focused on the effects of interfacial shear on the fibrillization kinetics of insulin. Human insulin served as a model protein for studying shear-induced fibrillization with relevance to amyloid diseases such as Alzheimer's, Parkinson's, prions, and type 2 diabetes. Insulin solutions at different protein concentrations were subjected to shear flows with prescribed interfacial angular velocities using a knife-edge (surface) viscometer (KEV) operating in a laminar axisymmetric flow regime where inertia is significant. Fibrillization kinetics were quantified using intrinsic fibrillization rate and times (onset, half, and end) determined through spectroscopic measurement of monomer extinction curves and fitting to a sigmoidal function. Additionally, the occurrence of gelation was determined through macroscopic imaging and transient fibril microstructure was captured using fluorescence microscopy. The results showed that increasing interfacial shear rate produced a monotonic increase in intrinsic fibrillization rate and a monotonic decrease in fibrillization time. Protein concentration did not significantly impact the intrinsic fibrillization rate or times; however, a minimum fibril concentration for gelation was found. Protein microstructure showed increasing aggregation and plaque/cluster formation with time.

Growth of microorganisms in an interfacially driven space bioreactor analog.

Fluid bioreactors in microgravity environments may utilize alternative methods of containment and mixing. The ring-sheared drop (RSD) is a containerless mixing device which functions in microgravity using surface tension for containment and mixes through interfacially-driven flow. To assess the feasibility of using interfacially driven flow devices, such as the RSD, as bioreactors, Escherichia coli growth and recombinant protein expression were analyzed in a ground-based analog of the RSD called the knife edge surface viscometer (KEV). Results demonstrated that the KEV can facilitate the growth of E. coli and that growth rate increases logarithmically with increasing knife edge rotation rate, similar to the standard growth method on Earth (orbital shaker). Furthermore, the KEV was shown to be viable for supporting recombinant protein expression in E. coli at levels comparable to those achieved using standard growth methods.