Recombinant Burkholderia pseudomallei UDP-3-O-acylglucosamine N-acyltransferase (lpxD) is produced in E.coli and represents the full-length protein from amino acids 1 to 361. This protein is engineered with an N-terminal 10xHis tag and a C-terminal Myc tag to aid in purification and detection. SDS-PAGE analysis confirms a purity of greater than 85%, which appears suitable for various research applications.
UDP-3-O-acylglucosamine N-acyltransferase, also known as lpxD, is an enzyme involved in the lipid A biosynthesis pathway - a critical component of the bacterial outer membrane. This enzyme catalyzes the transfer of an acyl group to UDP-3-O-acylglucosamine, an essential step in lipid A synthesis. Studying lpxD may provide insights into bacterial membrane assembly and could reveal potential antibiotic targets, though this remains an active area of investigation.
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.
Burkholderia pseudomallei lpxD is a bacterial enzyme involved in lipid A biosynthesis that requires precise folding and proper active site formation for its acyltransferase activity. The E. coli expression system is homologous to this bacterial protein, significantly increasing the probability of correct folding. However, the dual N-terminal 10xHis-tag and C-terminal Myc-tag may sterically interfere with the protein's functional domains or oligomerization interfaces. While the homologous system provides favorable folding conditions, experimental validation remains essential to confirm structural integrity and enzymatic activity.
1. Antibody Development and Immunoassay Studies
Antibody production depends on sequence availability rather than functional folding. If correctly folded (verified), the protein excels for generating conformation-sensitive antibodies. If misfolded/unverified, it remains highly suitable for producing antibodies against linear epitopes.
2. Protein-Protein Interaction Studies Using Tag-Assisted Pull-Down Assays
This application requires proper folding validation. Enzyme interactions within lipid A biosynthesis pathways require precise tertiary structure. If correctly folded (verified), the protein is suitable for identifying physiological interaction partners. If misfolded/unverified, there is high risk of non-specific binding or interaction failure.
3. Biochemical Characterization and Enzyme Kinetics Analysis
These studies are essential for determining folding status and functional competence. Functional characterization requires proper folding validation. If correctly folded and active (verified), characterization provides reliable data on enzymatic activity and kinetic parameters. If misfolded/inactive (unverified), analysis yields physical property data but enzymatic assays will not reflect native activity.
4. Comparative Structural and Functional Studies
Meaningful comparative studies require native protein conformation. If correctly folded (verified), the protein enables valid functional comparisons with homologs. If misfolded/unverified, comparative analyses would yield misleading evolutionary insights.
Final Recommendation & Action Plan
The homologous E. coli expression system provides favorable conditions for this bacterial enzyme, but experimental validation of structural integrity and enzymatic activity is essential before reliable use in functional studies. Begin with Application 3 (Biochemical Characterization) to assess folding quality through size-exclusion chromatography, circular dichroism spectroscopy, and validate enzymatic activity using appropriate acyltransferase assays. Once correct folding and functional activity are verified, proceed cautiously with Applications 2 and 4 for interaction studies and comparative analyses. Application 1 (antibody development) can proceed immediately regardless of folding status. If misfolding is detected, limit applications to linear epitope antibody production and basic biophysical characterization, avoiding all functional interaction and comparative studies.
