Journal
BIOCHEMISTRY
Volume 60, Issue 24, Pages 1876-1884Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.1c00138
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Funding
- National Institutes of Health [P41 GM128577, GM120582, AI116119, AI140541]
- Ohio Eminent Scholar funds
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Protein overexpression and purification are critical for in vitro studies, but can be challenging for some proteins. A new liquid chromatography-mass spectrometry method can help analyze target proteins in cell lysates. This approach may be valuable for optimizing large-scale protein production for structural biology and biotherapeutic research.
Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or proteinspecific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test 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 direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC-OBE-nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.
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