Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain

Autor: Anne Susemihl, Felix Nagel, Piotr Grabarczyk, Christian A. Schmidt, Mihaela Delcea
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: Molecules, Vol 26, Iss 24, p 7576 (2021)
Druh dokumentu: article
ISSN: 1420-3049
82484228
DOI: 10.3390/molecules26247576
Popis: Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.
Databáze: Directory of Open Access Journals
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