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Article

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer

1
NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079 Göttingen, Germany
2
Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
3
Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center, Von-Siebold-Straße 3a, 37075 Göttingen, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Nick Devoogdt
Biomolecules 2021, 11(2), 269; https://doi.org/10.3390/biom11020269
Received: 29 January 2021 / Revised: 8 February 2021 / Accepted: 9 February 2021 / Published: 12 February 2021
(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)
Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects. View Full-Text
Keywords: nanobody; sdAbs; epitope tag; affinity; immunoprecipitation; native elution; cold-elutable; synthetic library; ALFA system; ALFA Selector nanobody; sdAbs; epitope tag; affinity; immunoprecipitation; native elution; cold-elutable; synthetic library; ALFA system; ALFA Selector
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MDPI and ACS Style

Kilisch, M.; Götzke, H.; Gere-Becker, M.; Crauel, A.; Opazo, F.; Frey, S. Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer. Biomolecules 2021, 11, 269. https://doi.org/10.3390/biom11020269

AMA Style

Kilisch M, Götzke H, Gere-Becker M, Crauel A, Opazo F, Frey S. Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer. Biomolecules. 2021; 11(2):269. https://doi.org/10.3390/biom11020269

Chicago/Turabian Style

Kilisch, Markus, Hansjörg Götzke, Maja Gere-Becker, Alexander Crauel, Felipe Opazo, and Steffen Frey. 2021. "Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer" Biomolecules 11, no. 2: 269. https://doi.org/10.3390/biom11020269

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