Recombinant Schizosaccharomyces pombe Meiotically up-regulated gene 84 protein (mug84)

What is mug84 and what are its basic characteristics?

Mug84 (Meiotically up-regulated gene 84 protein) is a 195 amino acid protein encoded by the mug84 gene in Schizosaccharomyces pombe. The protein is characterized by its upregulation during meiotic processes in fission yeast. The full protein sequence is:

MTLTHHSTFIKGIEGSAEGEIEDVRQTTVFDPPFYGHPMLVPPSPSLTTMFRTRSTTPDE EGTAIAEIDQQDWDIMVKVPTYEYYGFVMYLVSMLGFGVYIVWALTPAPVLKFFEIHYYL SRWWALAIPTWLFVLVIYIHVVLNAYNTEVLTKPFSSLECIVDQYALVGEEDGAAHGRVV DLRLCDVNKQQLEET

The protein has a UniProt ID of O13904 and is also referenced under the synonyms mug84 and SPAC22A12.13 .

How is recombinant mug84 typically expressed and what expression systems are recommended?

Recombinant mug84 is typically expressed in E. coli expression systems with an N-terminal His tag for purification purposes. The full-length protein (amino acids 1-195) can be successfully expressed in bacterial systems, which suggests it does not have significant toxicity or insolubility issues that would prevent prokaryotic expression .

For researchers designing expression experiments:

• Use a bacterial expression vector containing a strong promoter

• Include an N-terminal His tag for affinity purification

• Express in standard E. coli strains optimized for recombinant protein production

• Consider temperature optimization during induction to maximize protein solubility

What purification and storage protocols are recommended for recombinant mug84?

The purified recombinant mug84 protein typically achieves >90% purity as determined by SDS-PAGE analysis . For optimal results:

Purification:

• Use affinity chromatography with Ni-NTA or similar matrices to capture the His-tagged protein

• Consider additional purification steps such as ion exchange or size exclusion chromatography if higher purity is required

• Elute in a Tris/PBS-based buffer system (pH 8.0)

Storage:

• Store the purified protein at -20°C/-80°C upon receipt

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Aliquot the protein for multiple uses to prevent degradation

• For short-term storage (up to one week), working aliquots may be kept at 4°C

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard) for long-term storage

• Create multiple small aliquots to minimize freeze-thaw cycles

What makes S. pombe a valuable model organism for studying meiotic proteins like mug84?

Schizosaccharomyces pombe (fission yeast) has emerged as a powerful tractable system for studying various cellular processes including meiosis and DNA damage repair. Key advantages include:

• S. pombe diverged from Saccharomyces cerevisiae approximately a billion years ago but shows higher conservation in chromosome structure and function genes with humans

• Many genes in S. pombe show similarity to genes involved in human disease, making it relevant for translational research

• The organism has well-characterized genetic and molecular tools optimized for studying DNA damage repair and recombination

• S. pombe centromeres are characterized by repetitive elements resembling higher metazoans, unlike the point centromeres in S. cerevisiae

• The fission yeast maintains stable chromosomal structures that facilitate the study of meiotic processes

These characteristics make S. pombe particularly valuable for studying meiosis-related proteins like mug84, which may have conserved functions across species.

What experimental approaches can be used to study mug84 function in S. pombe?

Several experimental approaches can be employed to study mug84 function:

Gene expression analysis:

• Monitor mug84 expression levels during different stages of meiosis using RT-qPCR

• Use RNA-seq to examine transcriptional profiles during meiotic progression

• Compare expression patterns in wild-type versus meiotic mutant strains

Functional analysis:

• Create gene knockouts or site-directed mutants to evaluate the functional consequences

• Implement the transcriptional induction systems available in S. pombe, such as the urg1 promoter system that allows induction within 30 minutes (faster than the traditional nmt1 promoter which requires 14-20 hours)

• Use chromosome loss assays and recombination assays to assess potential roles in genome stability

Protein localization:

• Generate fluorescently tagged versions of mug84 to track subcellular localization

• Implement LacO arrays as described in the literature to monitor interactions with specific chromosomal regions

• Perform chromatin immunoprecipitation (ChIP) to identify DNA binding sites if mug84 interacts with DNA

What methods are available to study protein-protein interactions involving mug84?

Several methodologies can be employed to study potential interaction partners of mug84:

In vitro methods:

• Pull-down assays using purified recombinant His-tagged mug84 as bait

• Surface Plasmon Resonance (SPR) to measure binding kinetics with candidate interactors

• Isothermal Titration Calorimetry (ITC) for quantitative binding analysis

In vivo methods:

• Yeast two-hybrid screening to identify novel interaction partners

• Co-immunoprecipitation experiments followed by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in live cells

• Fluorescence Resonance Energy Transfer (FRET) to study proximity of tagged proteins

Genetic approaches:

• Synthetic genetic interaction screens to identify functional relationships

• Suppressor screens to identify genes that compensate for mug84 mutations

• Epistasis analysis to place mug84 in known meiotic or DNA repair pathways

How might mug84 be involved in DNA damage repair pathways in S. pombe?

While the specific role of mug84 in DNA damage repair is not explicitly detailed in the search results, several approaches can be used to investigate potential functions based on established S. pombe assays:

Recombination assays:

• Implement the non-tandem repeat assays described by Schuchert and Kohli to study potential roles in crossover frequency

• Utilize direct repeat systems to examine potential functions in single-strand annealing (SSA) or break-induced replication (BIR)

• Apply RTS1-based replication fork stalling systems to assess potential roles in replication fork restart

DNA damage sensitivity:

• Evaluate sensitivity of mug84 mutants to various DNA damaging agents (e.g., MMS, HU, UV, IR)

• Combine mug84 mutations with established DNA repair pathway mutations to identify genetic interactions

• Assess chromosome loss rates in mug84 mutants using chromosome loss assays like Ch16-LMYAU

Protein recruitment analysis:

• Track the recruitment kinetics of mug84 to induced DNA breaks

• Compare recruitment patterns with established DNA repair factors

• Examine how mug84 localization changes in response to different types of DNA damage

What considerations should researchers keep in mind when designing experiments using recombinant mug84?

Researchers working with recombinant mug84 should consider several technical aspects:

Protein quality assessment:

• Verify proper folding using circular dichroism or thermal shift assays

• Assess aggregation status using dynamic light scattering

• Consider performing limited proteolysis to identify stable domains

Experimental controls:

• Include appropriate negative controls (e.g., unrelated His-tagged proteins) in binding experiments

• Use catalytically inactive mutants as negative controls in functional assays

• Consider using untagged protein preparations to rule out tag interference

Buffer optimization:

• Test multiple buffer conditions to maximize protein stability

• Evaluate the addition of stabilizing agents like glycerol or specific salt concentrations

• Consider the effect of pH on protein stability and activity

Storage considerations:

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Store aliquots at -80°C for long-term preservation

• Consider adding protease inhibitors to prevent degradation during storage

How can researchers integrate studies of mug84 into broader investigations of meiotic processes?

To position mug84 research within the broader context of meiotic studies:

Temporal expression analysis:

• Map mug84 expression relative to other meiotic markers to identify its precise timing during meiosis

• Use synchronized meiotic cultures to determine when mug84 is most active

• Correlate expression patterns with specific meiotic events (pairing, synapsis, recombination)

Genetic interaction mapping:

• Cross mug84 mutants with strains carrying mutations in known meiotic regulators

• Use synthetic genetic arrays to systematically identify genetic interactions

• Implement high-throughput screening approaches to place mug84 in known pathways

Functional complementation:

• Test whether mug84 homologs from other species can complement S. pombe mug84 mutants

• Identify conserved versus species-specific functions through domain swapping experiments

• Explore potential roles in higher eukaryotes through heterologous expression studies

What challenges might researchers encounter when working with recombinant mug84 and how can they be addressed?

Common challenges and solutions include:

Solubility issues:

• If initial expression yields insoluble protein, optimize induction conditions (lower temperature, reduced IPTG concentration)

• Consider fusion tags beyond His-tag (e.g., MBP, SUMO) that can enhance solubility

• Explore different E. coli strains optimized for difficult-to-express proteins

• Test co-expression with chaperones to improve folding

Stability concerns:

• Monitor protein stability over time using analytical techniques like size exclusion chromatography

• Identify optimal buffer conditions that maximize stability

• Consider adding stabilizing agents like glycerol or specific binding partners

• Use experimental techniques compatible with the protein's stability window

Activity verification:

• Develop activity assays based on predicted function (if known)

• Compare activity of freshly purified protein versus stored samples

• Assess the impact of tags on activity by comparing tagged versus untagged versions

How can researchers effectively troubleshoot experimental issues with S. pombe meiotic protein studies?

When troubleshooting experiments involving meiotic proteins in S. pombe:

Expression verification:

• Confirm mug84 expression using Western blotting with antibodies against the protein or its tag

• Use RT-qPCR to verify transcriptional upregulation during meiosis

• Implement proteomics approaches to monitor protein levels during meiotic progression

Localization problems:

• If fluorescently tagged proteins show diffuse localization, optimize fixation protocols

• Test different linker lengths between the protein and fluorescent tag

• Consider alternative tagging strategies (N-terminal versus C-terminal) to minimize interference

Functional redundancy:

• Create double or triple mutants to address potential redundancy with related proteins

• Use conditional alleles to bypass essential functions while studying meiotic roles

• Implement RNA interference or degron approaches for acute protein depletion

What analytical approaches are recommended for interpreting results from mug84 studies?

For robust data analysis and interpretation:

Quantitative analysis:

• Use appropriate statistical methods for comparing wild-type versus mutant phenotypes

• Implement multiple biological and technical replicates to ensure reproducibility

• Consider power analysis to determine appropriate sample sizes

Comparative genomics:

• Compare mug84 sequence, structure, and function across fungal species

• Identify conserved domains that might indicate functional importance

• Use phylogenetic analysis to trace evolutionary relationships of mug84 homologs

Integrative analysis:

• Combine results from multiple experimental approaches (genetic, biochemical, cell biological)

• Integrate mug84 data with existing datasets on meiotic processes in S. pombe

• Use computational modeling to generate testable hypotheses about mug84 function

How should contradictory results in mug84 research be approached and resolved?

When facing contradictory results:

Experimental validation:

• Replicate experiments under identical conditions to verify reproducibility

• Modify key parameters systematically to identify variables affecting outcomes

• Implement alternative methodologies to approach the question from different angles

Critical evaluation:

• Assess potential confounding factors in experimental design

• Consider strain background differences that might influence results

• Evaluate whether tags or fusion constructs might alter native protein function

Collaborative resolution:

• Engage with other laboratories studying related processes

• Consider sending materials for testing in different laboratory environments

• Design definitive experiments that can specifically address contradictions