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.
1. Antibody Development and Immunoassay Studies
This recombinant lpxD protein can serve as an immunogen for generating polyclonal or monoclonal antibodies specific to Burkholderia pseudomallei UDP-3-O-acylglucosamine N-acyltransferase. The dual tagging system offers versatile detection and purification strategies during antibody screening and validation processes. While the 85% purity level is generally sufficient for immunization protocols, researchers should consider whether additional purification steps might be beneficial for their specific antibody characterization studies. Such antibodies could become valuable research tools for examining lpxD expression patterns and cellular localization in bacterial systems.
2. Protein-Protein Interaction Studies Using Tag-Assisted Pull-Down Assays
The N-terminal His-tag and C-terminal Myc-tag make this protein well-suited for investigating potential protein interactions within the lipid A biosynthesis pathway. His-tag mediated pull-down experiments using nickel-affinity resins can capture lpxD along with any associated proteins from bacterial lysates. The Myc-tag provides an additional handle for immunoprecipitation studies or Western blot detection of the protein complex. These interaction studies might reveal novel regulatory mechanisms or pathway components involved in lipopolysaccharide biosynthesis, though controls will be essential to distinguish specific interactions from non-specific binding.
3. Biochemical Characterization and Enzyme Kinetics Analysis
Although biological activity has not been tested, this recombinant protein can be used for comprehensive biochemical characterization studies. These include protein stability, optimal buffer conditions, and thermal properties. The protein can serve as a starting point for developing and optimizing enzymatic activity assays using appropriate cofactors and substrates typical for N-acyltransferases. Initial screening experiments could establish baseline conditions for future activity measurements, though researchers should be aware that recombinant proteins sometimes exhibit different properties compared to their native counterparts.
4. Comparative Structural and Functional Studies
This full-length recombinant lpxD protein enables comparative analysis with homologous N-acyltransferases from other bacterial species to understand evolutionary relationships and functional conservation. Limited proteolysis experiments could help identify stable domains and potential functional regions. Cross-reactivity studies with antibodies or binding partners from related bacterial species may provide insights into conserved epitopes and functional domains within the lpxD family of enzymes, although such conservation patterns can sometimes be misleading without additional structural evidence.
