Recombinant UPF0234 protein MAP_4063c (MAP_4063c)

What is UPF0234 protein MAP_4063c and what are its key characteristics?

UPF0234 proteins belong to a family of bacterial proteins with conserved structure but often uncharacterized function (hence the UPF designation - Uncharacterized Protein Family). MAP_4063c specifically refers to the UPF0234 protein from Mycobacterium avium subsp. paratuberculosis. Structurally, UPF0234 proteins demonstrate high confidence prediction scores when analyzed through computational methods such as AlphaFold, with global pLDDT scores often above 90, indicating reliable structural models despite limited experimental validation .

The protein typically contains approximately 160-165 amino acids, based on sequence data from related UPF0234 family members such as plu3881 from Photorhabdus laumondii, which has 163 amino acids . UPF0234 proteins appear to be conserved across various bacterial species, including Escherichia coli (YajQ), Photorhabdus laumondii, and Mycobacterium species, suggesting potentially important cellular functions.

Which expression systems are most suitable for producing recombinant UPF0234 proteins?

Multiple expression systems have been successfully employed for UPF0234 protein production, with E. coli being the most commonly utilized. Based on available expression system data, the following options are viable for UPF0234 protein expression:

For most applications involving UPF0234 proteins, E. coli expression systems have demonstrated sufficient efficacy, providing good yields of soluble protein . The BL21-AI E. coli strain has been specifically designed for recombinant protein expression from T7-based expression vectors, offering a potential advantage for expression of challenging proteins .

How can I optimize experimental conditions for soluble expression of UPF0234 proteins?

Optimizing soluble expression requires a systematic approach to experimental design. For UPF0234 proteins, consider implementing a multivariant analysis approach that evaluates multiple parameters simultaneously:

• Implement statistical experimental design: Use factorial designs to systematically evaluate the impact of multiple variables on protein expression. This approach is more efficient than the traditional univariant method and allows for the characterization of experimental error while gathering high-quality information with fewer experiments .

• Key variables to optimize:

  • Induction temperature (typically testing 15°C, 25°C, and 37°C)

  • Inducer concentration

  • Media composition

  • Cell density at induction (OD600)

  • Induction duration (with evidence suggesting that 4-6 hours can be optimal for many proteins)

• Expression time considerations: Research indicates that induction times longer than 6 hours can be associated with lower productivity for some proteins, while the 4-6 hour range often presents similar productivity levels. For metabolic efficiency, selecting the shortest effective induction time (e.g., 4 hours) can maximize productivity while minimizing operational time .

What purification strategies are most effective for UPF0234 proteins?

While specific protocols for MAP_4063c purification are not detailed in the available literature, general approaches for similar bacterial proteins can be applied:

• Tag selection: For UPF0234 proteins, affinity tags such as His-tag or GST-tag can facilitate purification. Commercial options offer UPF0234 proteins with various tag configurations, including Avi-tag biotinylated versions that allow highly specific streptavidin-based purification .

• Purification workflow:

  • Initial capture using affinity chromatography (based on the incorporated tag)

  • Intermediate purification using ion exchange chromatography

  • Polishing step using size exclusion chromatography if necessary

• Tag removal considerations: If the presence of a tag might interfere with functional studies, incorporate a protease cleavage site between the tag and the protein of interest.

• Quality assessment: Target approximately 75% homogeneity after purification, which has been demonstrated as achievable for other recombinant bacterial proteins while retaining functional activity .

What experimental design approaches can maximize yield and functionality of recombinant UPF0234 proteins?

Advanced experimental design techniques can significantly enhance both expression yield and protein functionality. For UPF0234 proteins, implementing a robust Design of Experiment (DoE) approach is recommended:

Research demonstrates that this systematic approach can yield significant improvements, with documented cases achieving up to 250 mg/L of soluble, functional recombinant protein in E. coli systems .

How does the structure of UPF0234 protein MAP_4063c compare to other UPF0234 family members?

Structural analysis provides important insights into potential functions and evolutionary relationships. Computational modeling data suggests:

What approaches should be used to troubleshoot expression issues specific to UPF0234 proteins?

When encountering expression challenges with UPF0234 proteins, a systematic troubleshooting approach is essential:

• Solubility challenges:

  • If inclusion bodies form, adjust induction temperature (reducing to 15-20°C)

  • Test co-expression with molecular chaperones

  • Evaluate the impact of fusion tags on solubility

  • Consider solubility enhancers in the buffer system

• Expression level optimization:

  • For low expression, assess codon optimization for the host organism

  • Evaluate different promoter systems

  • Test multiple E. coli strains, including BL21-AI which is specifically designed for recombinant protein expression from T7-based vectors

• Systematic parameter adjustment:

  • When multiple parameters require optimization, use a structured DoE approach rather than changing one variable at a time

  • This statistical approach enables more thorough analysis and characterization of experimental error while requiring fewer experiments

What methods are recommended for functional characterization of UPF0234 proteins?

Given the often unknown function of UPF0234 family proteins, comprehensive functional characterization requires a multi-faceted approach:

• Biochemical characterization:

  • Evaluate nucleotide binding capacity (ATP, GTP)

  • Test RNA binding capabilities

  • Assess protein-protein interactions through pull-down assays

  • Determine oligomerization state through size exclusion chromatography

• Structural analysis:

  • If wet-lab structural determination is challenging, leverage computational models

  • AlphaFold models of UPF0234 proteins show high confidence scores (pLDDT >90), suggesting reliable structure prediction despite limited experimental validation

  • Use structural information to identify potential active sites or binding pockets

• Functional genomics approaches:

  • Consider gene knockout studies in native organisms

  • Evaluate phenotypic changes in various growth conditions

  • Employ RNA-Seq to identify transcriptional changes associated with the protein

How can advanced analytical techniques be applied to study UPF0234 protein interactions?

Understanding protein interactions is crucial for elucidating biological function. For UPF0234 proteins, consider these advanced analytical approaches:

• Protein-protein interaction analysis:

  • Bait-prey systems using biotinylated versions of the protein (Avi-tag technology allows site-specific biotinylation)

  • Proximity-based labeling techniques (BioID, APEX)

  • Mass spectrometry-based interactome mapping

• Structural biology approaches:

  • While computational models provide a starting point (with high confidence scores >90 pLDDT for UPF0234 family proteins), experimental validation through X-ray crystallography or cryo-EM would provide higher resolution details

  • NMR studies for dynamic regions or smaller domains

• Systems biology integration:

  • Network analysis to position UPF0234 within cellular pathways

  • Correlation analysis with other proteins of known function

  • Evolutionary analysis across bacterial species to identify conserved interaction partners