Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

Abstract

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn-tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76%. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90%). Furthermore, this “reaction-ready” system demonstrated excellent stability during five days of continuous-flow. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens new opportunities for continuous flow biosynthesis.

Vortex fluidic mediated encapsulation of functional fish oil featuring in situ probed small angle neutron scattering

“…Enzymatic reactions in the VFD have on average a sevenfold acceleration in their catalytic reactions 9 , depending on the choice of processing parameters of the device, including rotation speed of the inclined glass tube 9 . Tethering enzymes to the surface of the glass tube has also been achieved for the synthesis of complex molecules in a single pass under continuous flow conditions 10 . Fabricating nanomaterials in the VFD has yielded unexpected results, also under continuous flow, for example, in assembling fullerene C 60 molecules into nano-tubules with hollow diameters of 100-400 nm, in the absence of a surfactant and without the need for further downstream processing 11 .…”

Continuous Flow Organophosphorus Chemistry

“…Reproduced from ref. [58] . nucleosides 34 were efficiently produced at the mg-scale and purified using the semi-preparative mode of the on-line HPLC (Figure 11(b)) in good to high yields starting from inosine (30) as sugar donor.…”

“…Innovative systems such as a vortex fluidic device (VFD) were also used for cascade biocatalyzed flow reactions. [58] An example by Raston, Weiss and co-workers featured enzymes that were immobilized through to their polyhistidine-tag to an IMAC resin charged with Ni 2+ cations located in a VFD (Figure 12(a)). A two-step enzymatic synthesis was next utilized to assess the efficiency of the system: the procedure featured a phosphodiesterase and an alkaline phosphatase allowing the conversion of bis(p-nitrophenol)phosphate monosodium salt into p-nitrophenoxide and p-nitrophenol phosphate, the latter being finally converted into p-nitrophenoxide and inorganic phosphate (Figure 12(b)).…”

Vortex Fluidic Chemical Transformations

“…30, 32] Using fused His n -tags proteins for bioconjugation with immobilized metal affinity chromatography (IMAC) resin allowed ten-minute purification and creation of a “reactor ready” system in a single continuous flow process. This proof of concept allowed six different proteins to be purified from complex cell lysate, including tobacco epi-aristolochene synthase (TEAS), a difficult, yet valuable biocatalyst.…”