Recombinant Pseudomonas putida Metapyrocatechase (xylE) represents a full-length protein expressed in E. coli, spanning 1-307 amino acids. The protein carries an N-terminal 10xHis-SUMO tag and a C-terminal Myc tag, which appear to simplify purification and detection processes. SDS-PAGE analysis suggests purity levels exceeding 90%, making it potentially suitable for research applications that demand high-quality protein preparations.
Metapyrocatechase functions as an enzyme in aromatic compound breakdown. It seems to play an important role in catechol's catabolic pathway. The enzyme catalyzes the oxidative cleavage of catechol to 2-hydroxymuconic semialdehyde, which may prove fundamental for bioremediation studies and research examining how microbes degrade environmental pollutants.
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.
1. Protein-Protein Interaction Studies Using Dual-Tag System
The N-terminal His-SUMO tag paired with the C-terminal Myc tag appears to offer valuable tools for exploring protein interactions through co-immunoprecipitation and pull-down assays. Metal affinity chromatography-based pull-downs using the His tag might help identify potential binding partners from bacterial lysates or purified protein libraries. Meanwhile, the Myc tag could prove useful for immunoprecipitation experiments with anti-Myc antibodies to capture protein complexes. This dual-tag approach likely allows researchers to validate interactions through different methods, potentially reducing false-positive results in protein interaction screens.
2. Antibody Development and Validation Platform
Recombinant metapyrocatechase may serve as an effective immunogen for producing both polyclonal and monoclonal antibodies specific to this enzyme. The >90% purity level appears adequate for immunization protocols and subsequent antibody characterization steps. Both Myc and His tags offer built-in controls for testing antibody specificity through competitive binding assays and cross-reactivity studies. Researchers could validate generated antibodies using Western blot, ELISA, and immunofluorescence techniques, with the tagged protein acting as a positive control.
3. Biochemical Characterization and Enzyme Kinetics Studies
This purified full-length protein (1-307aa) likely provides an excellent foundation for thorough biochemical analysis. Studies might include thermal stability assessments, pH optimization, and determining cofactor requirements. Size exclusion chromatography and dynamic light scattering experiments could reveal the enzyme's oligomerization state and solution behavior patterns. The high purity level should enable precise protein concentration measurements for quantitative biochemical assays and comparative studies with other catechol-cleaving enzymes from various bacterial species.
4. Tag-Assisted Protein Purification Method Development
The dual-tag system makes this protein an attractive model for developing and refining multi-step purification protocols that combine metal affinity chromatography with immunoaffinity techniques. Researchers might use this protein to establish standardized protocols for His-SUMO tag cleavage and subsequent purification steps. The protein could serve as a benchmark for evaluating how efficiently different tag removal strategies work and comparing yields across various purification approaches for similarly tagged recombinant proteins.
