Recombinant Human Friend leukemia integration 1 transcription factor (FLI1) is expressed in E. coli and contains amino acids 1-452, representing the full-length protein. The protein includes an N-terminal 10xHis tag to aid in purification, with a purity level above 85% as determined by SDS-PAGE. This product is for research use only and should not be used in diagnostic or therapeutic applications.
FLI1 is a transcription factor that regulates gene expression and appears to play a crucial role in blood cell development and vascular tissue formation. It belongs to the ETS family of transcription factors, which bind to specific DNA sequences and influence various cellular pathways. FLI1 has gained attention in research primarily because of its involvement in cellular differentiation and proliferation.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
The E. coli system lacks eukaryotic chaperones and post-translational modification machinery, which are critical for the proper folding of complex human transcription factors like FLI1, particularly its ETS DNA-binding domain and PNT domain required for functional activity (e.g., DNA binding and transcriptional activation). The N-terminal His-tag may sterically interfere with the N-terminal region of FLI1, potentially disrupting its native conformation or oligomerization state. While full-length coverage increases the probability of containing all functional domains, the absence of validation data (e.g., circular dichroism for secondary structure, size-exclusion chromatography for oligomeric state, or DNA-binding assays) means the protein cannot be assumed to be correctly folded or bioactive. Experimental verification is essential before any functional application.
1. Protein-DNA Interaction Studies
This recombinant FLI1 protein could be used to study DNA-binding properties only if its folding is experimentally verified. The full-length sequence contains the ETS DNA-binding domain, and the His-tag facilitates immobilization for techniques like EMSA or SPR. However, if the protein is misfolded, its DNA-binding interface may be altered, leading to inaccurate measurements of binding affinity or specificity (e.g., false negatives in recognizing ETS-binding sites). The N-terminal tag could potentially interfere with DNA interaction if it disrupts the native N-terminal structure. Any results require validation using native FLI1 from mammalian cells or functional assays.
2. Antibody Development and Validation
The recombinant FLI1 protein is suitable as an immunogen for generating antibodies targeting linear epitopes, as antibody production primarily relies on amino acid sequences rather than native conformation. The high purity (>85%) reduces contamination risks. However, if antibodies are intended to recognize conformational epitopes of native FLI1 (e.g., in chromatin immunoprecipitation or immunofluorescence), misfolding of the recombinant protein may result in antibodies that fail to bind the functional transcription factor in its physiological context. The His-tag could also induce tag-specific antibodies, requiring careful screening.
3. Protein-Protein Interaction Analysis
The His-tagged FLI1 can be used technically in pull-down assays to screen for interaction partners (e.g., transcriptional co-factors). However, the biological relevance of interactions depends entirely on correct folding. Misfolding may expose non-physiological binding surfaces, leading to false-positive interactions or failure to capture genuine partners (e.g., components of transcriptional complexes). The full-length design is advantageous, but any identified interactions must be cross-validated with native FLI1 from mammalian systems or using complementary methods like co-immunoprecipitation of endogenous proteins.
4. Structural and Biophysical Characterization
The protein’s suitability for structural studies (e.g., X-ray crystallography) is strictly dependent on proper folding and homogeneity. Techniques like dynamic light scattering or analytical ultracentrifugation could be applied, but data from a misfolded protein would not reflect the native structure of FLI1. The His-tag and E. coli expression system may promote aggregation or non-native oligomerization, complicating interpretation. Prior validation of folding (e.g., via circular dichroism or SEC-MALS) is a prerequisite for meaningful structural insights.
5. In Vitro Transcription Assays
This application is fully contingent on confirmed bioactivity. FLI1’s role as a transcription factor requires precise folding for DNA binding and transcriptional activation. If the recombinant protein is misfolded or inactive, it would fail to regulate transcription in cell-free systems, leading to misleading results about its regulatory mechanisms. The protein must first be validated in a functional assay (e.g., DNA-binding or reporter gene activation) before use in transcriptional studies.
Final Recommendation & Action Plan
To ensure reliable outcomes, prioritize experimental validation of the protein’s folding and bioactivity before functional applications. Begin with biophysical characterization using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state and monodispersity, and circular dichroism spectroscopy to evaluate secondary structure content against known FLI1 spectra. Then, perform functional assays such as an electrophoretic mobility shift assay (EMSA) with a known ETS-binding site DNA sequence to confirm DNA-binding capability, or a cell-based reporter assay if feasible. If validation succeeds, the protein can be cautiously used for the proposed applications, with disclosures about tag-related limitations; if validation fails, restrict use to non-conformation-dependent applications like linear-epitope antibody production.
