Recombinant Saccharomyces cerevisiae Alpha-1,3-mannosyltransferase MNT3 is produced using the baculovirus expression system, covering the amino acid region 32-630. This partial protein includes an N-terminal 10xHis-tag, which helps with purification and detection. SDS-PAGE analysis confirms the protein maintains a purity level greater than 85%, making it suitable for various research applications that require high-quality reagents.
Alpha-1,3-mannosyltransferase MNT3 appears to be an enzyme involved in glycosylation processes within Saccharomyces cerevisiae. It plays what seems to be a critical role in modifying glycoproteins by adding mannose residues—a key step in synthesizing complex carbohydrates. This protein may offer valuable insights into understanding how glycan assembly works and how it's regulated, potentially shedding light on cellular processes and pathways that glycosylation influences.
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 Mannosyltransferase Activity Assays
This recombinant MNT3 protein can help establish and optimize in vitro enzymatic assays for studying α-1,3-mannosyltransferase activity using synthetic or natural acceptor substrates. The N-terminal His-tag makes protein purification easier and helps with immobilization for enzyme kinetics studies. Researchers might investigate substrate specificity, optimal reaction conditions, and kinetic parameters of this yeast mannosyltransferase. The baculovirus expression system typically produces proteins with appropriate post-translational modifications that could be important for enzymatic function.
2. Glycosylation Pathway Reconstitution Studies
The purified MNT3 protein can be incorporated into cell-free glycosylation systems to study mannan biosynthesis pathways in yeast. This approach allows researchers to break down the sequential steps of N-linked and O-linked glycosylation by combining MNT3 with other purified glycosyltransferases and appropriate donor/acceptor molecules. A controlled in vitro environment enables detailed analysis of glycan structure formation and helps determine MNT3's specific contribution to yeast cell wall mannoprotein biosynthesis.
3. Antibody Development and Immunological Studies
The His-tagged recombinant MNT3 protein serves as an antigen for generating specific antibodies against this mannosyltransferase. These antibodies can then be used in subsequent research applications including Western blotting, immunoprecipitation, and immunofluorescence studies of native MNT3 in yeast cells. The high purity level (>85%) should minimize cross-reactivity with other proteins during antibody production and validation.
4. Protein-Protein Interaction Studies
The N-terminal His-tag makes pull-down assays and affinity chromatography experiments possible to identify potential protein partners that interact with MNT3 in the yeast secretory pathway. This recombinant protein can work as bait in biochemical interaction studies with yeast cell lysates or other purified proteins involved in glycosylation processes. Such studies may reveal regulatory mechanisms or protein complexes involved in mannosyltransferase function and localization.
5. Structural and Biophysical Characterization
The purified MNT3 protein provides material for structural biology approaches including X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy studies. Biophysical techniques such as dynamic light scattering, analytical ultracentrifugation, and thermal stability assays can be performed to characterize the protein's oligomerization state, stability, and conformational properties. The baculovirus expression system often yields protein suitable for structural studies due to proper protein folding, though success isn't guaranteed.
