Recombinant Brugia malayi tRNA (guanine-N(7)-)-methyltransferase (Bm1_01445) is expressed in E.coli, covering amino acids 1 to 258, and is provided as a full-length protein. It features an N-terminal 10xHis-tag for ease of purification and detection. The protein is purified to a level greater than 85% as determined by SDS-PAGE, ensuring a high-quality reagent suitable for various research applications.
tRNA (guanine-N(7)-)-methyltransferase is an enzyme involved in the post-transcriptional modification of tRNA molecules. This protein catalyzes the methylation of guanine bases, a critical step in tRNA maturation that influences the stability and function of tRNA. Studying this enzyme appears important for understanding the molecular mechanisms underlying RNA modification processes, which seem essential in the regulation of gene expression.
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. In Vitro Enzymatic Activity Characterization
This recombinant tRNA methyltransferase can be used to set up and optimize enzymatic assays for measuring guanine-N(7)-methylation activity on tRNA substrates. The purified protein allows for controlled biochemical studies to determine substrate specificity, kinetic parameters, and cofactor requirements under defined conditions. Such characterization would likely provide fundamental insights into the catalytic mechanism of this filarial nematode enzyme. The N-terminal His-tag makes protein purification and immobilization simpler for various assay formats.
2. Comparative Enzyme Studies Between Species
The recombinant Brugia malayi methyltransferase can serve as a reference enzyme for comparative biochemical studies with orthologous tRNA methyltransferases from other nematode species or model organisms. Researchers can examine differences in substrate preferences, catalytic efficiency, and structural requirements across species. This comparative approach would help reveal evolutionary relationships and functional divergence within the tRNA methyltransferase family. The standardized recombinant format ensures reproducible comparisons between different enzyme variants.
3. Antibody Development and Validation
The purified recombinant protein can be used as an immunogen for generating specific antibodies against the Brugia malayi tRNA methyltransferase. The His-tagged protein appears suitable for immunization protocols and subsequent antibody screening using ELISA or Western blot techniques. Generated antibodies could be validated for specificity using the same recombinant protein in various immunoassay formats. Such antibodies would likely be valuable research tools for studying the native enzyme in filarial nematode extracts or tissue samples.
4. Protein-Protein Interaction Studies
The His-tagged recombinant methyltransferase can be used in pull-down assays to identify potential protein partners or cofactors that interact with this enzyme in filarial nematodes. The affinity tag enables immobilization on nickel-based resins for capturing interacting proteins from nematode lysates or purified protein libraries. Mass spectrometry analysis of pulled-down complexes could reveal novel regulatory proteins or pathway components. This approach would contribute to understanding the cellular context and regulation of tRNA modification in parasitic nematodes.
5. Structural and Biophysical Analysis
The recombinant protein provides material for structural biology approaches including X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy studies. Biophysical characterization techniques such as dynamic light scattering, differential scanning calorimetry, or analytical ultracentrifugation can be performed to determine protein stability, oligomerization state, and thermal properties. Having purified protein available enables systematic structure-function relationship studies through site-directed mutagenesis experiments. Such structural insights would advance understanding of tRNA methyltransferase mechanisms in parasitic organisms.
