A High-Throughput Strategy for Recombinant Protein Expression and Solubility Screen in Escherichia coli : A Case of Sensor Histidine Kinase
| Autor: | Jan Hejátko, Blanka Pekárová, Agnieszka Szmitkowska |
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| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
Lysis biology Chemistry 030302 biochemistry & molecular biology Histidine kinase biology.organism_classification medicine.disease_cause law.invention 03 medical and health sciences Biochemistry law Lysis buffer medicine Protein biosynthesis Recombinant DNA Arabidopsis thaliana Escherichia coli Intracellular 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493998838 |
| Popis: | Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains. |
| Databáze: | OpenAIRE |
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