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Screening_tagged_proteins_using_tandem_affinity-buffer_exchange_chromatography_online_with_native_mass_spectrometry-1.pdf (3.76 MB)

Screening Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry

preprint
submitted on 15.02.2021, 15:58 and posted on 16.02.2021, 13:23 by Florian Busch, Zachary VanAernum, Stella M. Lai, Venkat Gopalan, Vicki Wysocki
Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express robustly in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to screen
multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography–mass spectrometry-based method for rapid, direct analysis of target proteins in cell lysates. This online approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based buffer exchange (OBE) and native mass spectrometry (nMS). The use of IMAC-OBE-nMS to optimize conditions for large-scale protein production should expedite structural biology and biotherapeutic initiatives.

Funding

Resource for Native Mass Spectrometry Guided Structural Biology

National Institute of General Medical Sciences

Find out more...

GM120582

AI116119

History

Email Address of Submitting Author

vanaernum.2@osu.edu

Institution

The Ohio State University

Country

United States of America

ORCID For Submitting Author

0000-0002-0956-3956

Declaration of Conflict of Interest

N/A

Version Notes

Version 1.0

Exports