Abstract
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.
Highlights
Introduction and Eduardo SommellaProteins with domains that participate in zinc ion (Zn2+ ) binding are widely represented across eukaryotic genomes
Introduction of the flexible peptide linker (GGGGS)3 is crucial for the subsequent Förster Resonance Energy Transfer (FRET) assays ensuring sufficient energy transfer
The flexibility of the fluorescent protein tag limits interference with the B-cell lymphoma/leukemia 11B (BCL11B)–BCL11B interaction
Summary
Introduction and Eduardo SommellaProteins with domains that participate in zinc ion (Zn2+ ) binding are widely represented across eukaryotic genomes. The most prominent zinc finger motif is the C2H2 coordination environment, where two cysteines and two histidines are involved in zinc binding [2] These typical C2H2 zinc fingers are best known for interacting with nucleic acids in a sequence-specific manner and, exerting critical functions in gene expression [3,4]. Based on these abilities, many zinc finger proteins are transcription factors (TFs). Many zinc finger proteins are transcription factors (TFs) Due to their role in several cellular processes, zinc finger proteins play key roles in various diseases, as the recent data highlights. Since they function as both oncogenes and tumor suppressor genes [5,6], altered expressions of specific zinc finger proteins is found, for example, in various human cancers, Parkinson’s disease, and congenital heart disease [5,7,8]
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