TY - JOUR
T1 - A nanobody-based microfluidic chip for fast and automated purification of protein complexes
AU - Keyser, Phebe De
AU - de Waard, Mitch
AU - Jimidar, Ignaas S. M.
AU - Verloy, Sandrien
AU - Janvier, Steven
AU - Kalichuk, Valentina
AU - Zögg, Thomas
AU - Wohlkönig, Alexandre
AU - Pardon, Els
AU - Steyaert, Jan
AU - Desmet, Gert
PY - 2024/11/26
Y1 - 2024/11/26
N2 - 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.
AB - 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.
KW - n/a OA procedure
UR - http://www.scopus.com/inward/record.url?scp=85210313674&partnerID=8YFLogxK
U2 - 10.1039/D4LC00728J
DO - 10.1039/D4LC00728J
M3 - Article
SN - 1473-0197
VL - 24
SP - 5421
EP - 5432
JO - Lab on a chip
JF - Lab on a chip
IS - 24
ER -