Recombinant Escherichia coli Transposase for transposon Tn5 (tnpA) is produced in an E. coli expression system and comes as a full-length protein without any tags. The expression region spans amino acids 1-476 and includes two specific mutations: E54K and L372P. SDS-PAGE analysis shows the protein achieves greater than 85% purity, which appears suitable for most research applications.
Transposase Tn5 represents a key enzyme in transposon movement, enabling DNA sequences to integrate into host genomes. Genetic engineering and functional genomics research rely heavily on this enzyme to investigate how genes work and how they're regulated. The enzyme's capacity to catalyze DNA rearrangements has made it indispensable for developing genetic assays and various biotechnology applications.
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.
Based on the provided information, the folding state and bioactivity of this Tn5 transposase (E54K, L372P) are unknown and must be considered unverified. The presence of two point mutations is a critical factor; these mutations may be designed to hyperactivate the enzyme, inactivate it, or alter its specificity. However, without functional validation, the protein's ability to perform transposition or bind DNA specifically cannot be assumed. The >85% purity indicates a preparation largely free of contaminants but provides no information on the correct tertiary structure or catalytic capability of the protein. Therefore, applications that require the protein's specific biological activity are highly speculative without experimental confirmation.
1. In Vitro Transposition Assay Development
This recombinant Tn5 transposase could be used in experiments aimed at developing transposition assays. However, the entire premise of this application is contingent on the unverified assumption that the protein is functional. The initial work should focus on rigorously testing for any transposition activity using gel electrophoresis or other methods. Until activity is confirmed, it cannot be said to be ""useful for establishing"" assays, but rather it is a candidate for such validation.
2. Protein-DNA Interaction Studies
The purified transposase can be used as a substrate for EMSA to investigate DNA-binding characteristics. However, a key caveat is that any observed binding must be shown to be specific. The mutations may abolish or alter specific DNA recognition. Therefore, experiments must include controls with non-specific DNA to distinguish specific binding from non-specific interactions. The comparison to wild-type transposase is a valid goal, but it requires that both proteins be correctly folded and retain at least some specific binding capability.
3. Biochemical Characterization of Effects
This double transposase is suitable for comparative biochemical and biophysical analysis, such as thermal stability assays, dynamic light scattering, and analytical ultracentrifugation. This application is valid and is independent of the protein's catalytic bioactivity, as it focuses on intrinsic physical properties. These studies can reveal how the mutations affect stability and oligomerization. It should be noted that any interpretation linking these biophysical changes to functional changes depends on subsequent activity assays.
4. Antibody Development and Validation
This recombinant Tn5 transposase is suitable for use as an antigen to generate antibodies against Tn5 transposase, specifically for detecting this variant. The high purity is adequate for immunization and antibody validation (e.g., Western blot, ELISA). This application is largely independent of the protein's native folding and bioactivity, as antibodies can be generated against linear epitopes.
Final Recommendation & Action Plan
The absolute first step must be to conduct a functional transposition assay to determine if this protein possesses any catalytic activity. The planned experiments for Application 1 (In Vitro Transposition Assay Development) are, in fact, this essential validation step and should be treated as such. The outcome of this activity test dictates the viability of Applications 1 and 2: a positive result would enable meaningful transposition and DNA-binding studies, while a negative result would limit the protein's utility to Applications 3 (biophysical characterization) and 4 (antibody production). Until bioactivity is confirmed, all proposals for functional studies should be considered preliminary and exploratory.
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