Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry.
Biochemistry
; 60(24): 1876-1884, 2021 06 22.
Article
em En
| MEDLINE
| ID: mdl-34100589
ABSTRACT
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 protein-specific (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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Proteínas
/
Cromatografia de Afinidade
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos