Shear‐Stress‐Mediated Refolding of Proteins from Aggregates and Inclusion Bodies

Abstract

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of the much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is >100-times faster than conventional, overnight dialysis. This rapid refolding technique could significantly shorten the times, lower costs, and reduce the waste streams associated with protein expression for a wide-range of industrial and research applications.

Organic oxidations promoted in vortex driven thin films under continuous flow

“…The conversion of thiols to disulfides is an important reaction in synthesis and biochemistry, with particular relevance to peptide and protein folding. 33,34 Given that the VFD has been explored as a technology for folding proteins in-flow, 26 the study of aerobic oxidations of cysteine to cystine also has broader relevance to bioorganic chemistry. As a starting point, a 1.0 mL solution of 1 in D 2 O was buffered to a pH of 9.8 and processed under a stream of oxygen both in a vial with simple stirring and also in the VFD in the confined mode of operation where a defined volume of liquid is placed in the tube at 45°tilt and rapidly rotated.…”

“…24 Reaction mixtures are rotated at high speeds (0-9k rpm), producing a large surface area as the liquid forms a thin film. The unique conditions found provide many benefits in organic, 25 biochemical 26,27 and materials chemistry. 28 Given the versatility of the VFD and its ease of use, we were motivated to explore the ways in which it could be integrated into oxidations that align with the tenets of green chemistry.…”

“…First, we posited that the thin film processing would improve gas exchange between the reaction medium and the atmosphere, perhaps improving the rate of aerobic oxidations and providing a simple and efficient method for deploying gaseous reagents in synthesis. Second, we suspected that the intense micro-mixing in the VFD that results from extremely rapid rotation of the inclined reaction tube, typically 45°for optimum processing, [25][26][27][28] would enable efficient mixing of immiscible materials such as non-polar organic substrates and oxidants in aqueous solution. The problem of immiscibility limits the use of these aqueous oxidants, even in conventional flow reactors, as evident by the use of phase transfer catalysis to promote such reactions.…”

Fabrication of Nanofiber Filtration Membranes Using Polyethylene Terephthalate (PET): A Review

“…So related to wanted condition, one of these two modes would be selected. Some other narrative researches that have been performed with the help of VFD are involved bacteria and algal cells with graphene and magnetic polymer [78][79][80], protein folding [81], exfoliating single layered graphene and hexagonal Boron Nitride (h-BN) sheets from the bulk material [82], sol-gel synthesis of silica xero gel at room temperature and the incorporation of curcumin, [83] controlling the pore size and wall thickness of mesoporous silica [84,85], fabricating toroidal arrays of SWCNTs, [86] laterally 'slicing' CNTs, [87] accelerating enzymatic reaction, [88] probing the structure of self-organized systems [89,90], biodiesel catalysis [91] and many others. According to the extra capabilities of VFD as a various reaction media compared with Spinning Disc Processors (SDPs) or Rotating Tube Processors (RTPs), VFD will be more advantageous in reaction accomplishment [92].…”

Rapid protein immobilization for thin film continuous flow biocatalysis

“…Recently, our laboratories have focused on utilizing thin films to mediate protein folding, 37 biocatalysis 38 and molecular assembly line processes. 39 This involves processing in a vortex fluidic device (VFD) which confines reagents to a ≈250 μM thin film.…”