Vortex fluidic devices (VFDs) used with immobilized metal affinity chromatography (IMAC) have provided an effective, albeit cumbersome, way to separate His-tagged proteins. Researchers recently detailed a simplified approach that purified His-tagged proteins in a single step, in less than 10 minutes, including elution time.
VFDs create a thin, liquid film that significantly improves mass transport, which enhances the binding of the protein to the affinity capture ligands. Previously, “the process of coating the VFD tube with IMAC resin was tedious, and the IMAC resin coating was unstable,” according to a recent paper by Gregory Weiss, PhD, director of innovation, School of Physical Sciences, University of California, Irvine, and colleagues Xuan Luo, PhD, research associate, College of Science and Engineering, and Colin Raston, PhD, professor clean technology, both of Flinders Institute for Nanoscale Science and Technology at Flinders University in Australia.
Instead, their solution features a membrane insert that is coated with any of several IMAC resins. This membrane insert acts as a substrate for the IMAC resin and boosts its stability inside the VFD. An alternative, resin-free approach modifies the membrane insert with iminodiacetic acid (IDA) ligands.
In either the resin or resin-free instance, rapidly rolling the tube at a 45° angle thoroughly exposes the substrate to binding, washing, or elution buffers, for continuous flow protein purification.
“The target protein His-tagged GFP can be washed with buffer and eluted with a high concentration of imidazole, analogous to conventional IMAC protein purification,” they report.
The paper provides step-by-step instructions for preparing the resin-loaded membrane insert and the VFD, purifying His-tagged proteins using the VFD, and preparing the resin-free substrate. Both clarified and non-clarified lysate examples are included.
Wide-spread compatibility
“For biopharmaceutical manufacturing, the approach could be adapted for rapid purification using a different type of affinity chromatography,” Weiss tells GEN. “For example, resin or membrane surfaces could be bioconjugated to Protein A to purify antibodies in continuous flow.”
This VFD purification protocol is compatible with bead- or membrane-based purification systems and with both clarified and non-clarified lysates, the team notes.
Weiss’ VFD membrane insert method lost only 10% of the total protein using resin from one commercial manufacturer, and 21% using resin from a second supplier. In contrast, traditional column chromatography lost 54% of the total protein using resin from the first manufacturer and 87% using resin from the second supplier.
This study was conducted using clarified and non-clarified Escherichia coli lysate containing overexpressed enhanced green fluorescent protein fused to a His6-tag (eGFP-His6), but can be adapted easily for other proteins.
Weiss and colleagues strongly recommend verifying the success of column-based purification to confirm the presence and availability of the tag on the target protein. Then, to maximize capture, they recommend cycling the lysate through the VFD tube three or more times.
They also point out that the choice of membrane filter paper affects results. Filters with high hydroxyl residue tend to offer more ligand binding sites, increasing capture efficiency.
Thanks to the high flow-through within the VFD, dynamic binding can occur as well as high levels of protein purity, they conclude.
