A nanobody-based microfluidic chip for fast and automated purification of protein complexes

Phebe De Keyser, Mitch de Waard, Ignaas S. M. Jimidar, Sandrien Verloy, Steven Janvier, Valentina Kalichuk, Thomas Zögg, Alexandre Wohlkönig, Els Pardon, Jan Steyaert, Gert Desmet

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Many proteins, especially eukaryotic proteins, membrane proteins and protein complexes, are challenging to study because they are difficult to purify in their native state without disrupting the interactions with their partners. Hence, our lab developed a novel purification technique employing Nanobodies® (Nbs). This technique, called nanobody exchange chromatography (NANEX), utilises an immobilised low-affinity Nb to capture the target protein, which is subsequently eluted – along with its interaction partners – by introducing a high-affinity Nb. In line with the growing trend towards studying proteins in smaller sample sizes, the present study validates miniaturisation of NANEX in a packed bed microfluidic (μNANEX) chip. This μNANEX setup integrates up to five submicroliter silicon chips, enabling fully automated and reproducible purifications within minutes. Additionally, a digital twin model of the μNANEX column, which accurately predicts the effect of the reaction kinetics and mass transfer on the elution peaks, has been validated over a broad range of experimental conditions. The effectiveness of the method is demonstrated with Nbs binding to the green fluorescent protein (GFP), allowing streamlined purification of any GFP fusion protein from biological samples. Specifically, we used μNANEX to purify 0.1–1 μg of GFP-fused yeast proteins from 20 μL crude lysate and identified their interaction partners via mass spectrometry, showing that μNANEX purification preserves protein complexes.
Original languageEnglish
Pages (from-to)5421-5432
Number of pages12
JournalLab on a chip
Volume24
Issue number24
Early online date26 Nov 2024
DOIs
Publication statusPublished - 26 Nov 2024

Keywords

  • n/a OA procedure

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