Recombinant Human Serine/arginine-rich splicing factor 9 (SRSF9) is expressed in E.coli and contains a complete protein sequence spanning amino acids 1-221. An N-terminal GST tag has been added, which appears to improve both stability and purification efficiency. SDS-PAGE analysis indicates the product achieves purity levels exceeding 90%, making it well-suited for research applications that demand high-quality protein samples.
SRSF9 plays a crucial role in regulating pre-mRNA splicing—a fundamental step in gene expression. As a member of the serine/arginine-rich splicing factor family, this protein seems essential for spliceosome assembly and splice site selection. Its involvement in alternative splicing suggests SRSF9 may be particularly valuable for researchers investigating gene regulation and cellular expression patterns.
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.
Based on the provided information, the recombinant human SRSF9 is expressed in E. coli, a prokaryotic system that is generally unsuitable for producing functional eukaryotic RNA-binding proteins like SRSF9. SRSF9 requires precise folding, specific phosphorylation (by SR protein kinases), and proper arginine-serine (RS) domain conformation for its splicing regulatory activity. E. coli lacks the eukaryotic chaperones, phosphorylation machinery, and proper cellular environment necessary for correct folding and modification of this complex regulatory protein. The presence of a large N-terminal GST tag (~26 kDa) may significantly interfere with the native protein structure and RNA-binding capability. While the protein is full-length (1-221aa) with >90% purity, the expression system makes it highly likely to be misfolded, unphosphorylated, and inactive without experimental validation of its RNA-binding and splicing regulatory functions.
1. GST Pull-Down Assays for Protein-Protein Interaction Studies
The GST tag enables technical feasibility for pull-down experiments, but if SRSF9 is misfolded (as expected in E. coli), it will not interact physiologically with true binding partners (e.g., other splicing factors, RNA polymerase, or spliceosome components). The RS domain requires precise conformation and phosphorylation for specific interactions. Identified interactions could be non-physiological artifacts. This application should not be pursued without confirmation of proper folding and phosphorylation status.
2. Antibody Development and Validation
The recombinant SRSF9 can serve as an effective immunogen for generating antibodies that recognize linear epitopes, even if the protein is misfolded. The full-length sequence ensures broad epitope coverage. However, antibodies may not recognize phosphorylation-dependent or conformational epitopes of native, properly modified SRSF9 in human cells. Validation against endogenous SRSF9 from mammalian cells is essential.
3. In Vitro Binding Studies with RNA Substrates
This application is highly problematic without activity validation. If SRSF9 is misfolded and unphosphorylated, RNA-binding studies will not reflect biological specificity. SRSF9 requires proper RS domain conformation and phosphorylation for specific RNA recognition. EMSA or filter binding assays may show non-specific binding rather than physiological RNA interactions. This application requires prior demonstration of proper folding and specific RNA-binding capability.
4. Biochemical Characterization and Functional Domain Mapping
This application is well-suited for assessing the recombinant human SRSF9 protein itself. Techniques such as limited proteolysis, mass spectrometry, and biophysical analyses can characterize the properties of the recombinant human SRSF9 protein. However, results will reflect the E. coli-expressed, GST-tagged protein. They may not accurately represent native SRSF9's domain organization or structure-function relationships due to likely misfolding and lack of phosphorylation.
Final Recommendation & Action Plan
Given the high probability of misfolding in E. coli for this complex eukaryotic RNA-binding protein, we recommend first performing comprehensive validation: 1) Biophysical characterization (circular dichroism for secondary structure, analytical ultracentrifugation for oligomeric state) to assess folding quality; 2) Functional validation using known SRSF9 RNA targets and phosphorylation assays; 3) Comparison with SRSF9 from mammalian expression systems if possible. Antibody development can proceed immediately as the safest application. Avoid all functional studies (interactions, RNA-binding) until proper folding and phosphorylation are confirmed. For reliable SRSF9 research, obtain the protein from mammalian expression systems capable of proper folding and post-translational modifications. Always include appropriate controls, such as phosphorylated SRSF9 and validated RNA targets, in experiments.
