Recombinant Schizosaccharomyces pombe Uncharacterized zinc metalloprotease C354.09c (SPBC354.09c)

What is SPBC354.09c in Schizosaccharomyces pombe?

SPBC354.09c is classified as an uncharacterized zinc metalloprotease in fission yeast (Schizosaccharomyces pombe). According to gene deletion studies, it is categorized as a class Ib gene that is viable when deleted, indicating it is not essential for basic cellular functions under standard laboratory conditions . The full-length protein consists of 794 amino acids and has been assigned the UniProt ID O43023 .

What is known about the evolutionary conservation of SPBC354.09c?

Gene deletion studies have identified YPL176c as the ortholog of SPBC354.09c in Saccharomyces cerevisiae (budding yeast) . Interestingly, while SPBC354.09c deletion strains are viable in S. pombe, the corresponding gene in S. cerevisiae is also viable when deleted. This conservation between distantly related yeast species suggests an important but non-essential function that has been maintained throughout evolutionary divergence.

What is the amino acid sequence and predicted structural features of SPBC354.09c?

The complete 794-amino acid sequence of SPBC354.09c is:

MTDEKHVYVPPPKDPPSYEEVALHSALNNSAPPNDGEQNETSMEEMEIIEPPSEDSSRFP
LLRTKLAAIHEGWESACHSFEIRFASTFHRIPFQFLYLAVIATVIILASYYGYFDGVPAW
RSVHHYGEDVLLNYIKGCDISDTRQQVMTLSSIPHLAGTVGDSSLLQMIMNRLYYEKGTI
VDFREFYAYLNFPQLVSLSIDGDDSFHPSLIESYQVGGYDGVSIPTPATFGGSPSGFVNA
PLVYANRGRIEDFEWLVNSGIYVESSIVLVRANQSDFALATANAEKYNASAILIFEDTYL
TSLDNLNQVYPAGPYPSANSLYRGSVANHYYYVGDPLTPGWSAHEETNRISPKDANVLPS
IVSIPITFNDGIELLKRLQGHGHLVKDSNWCQDLAPVLSEVWTGSKISSPGLEVNVLQDI
EDKQKIINIMAQIDGYESDQILVVGAPRDSWCTGASDSSVGTSLLIDVISTFANMAQDLS
WKPRRTIVFASWDARQFNAIGSTEFLEYWKESLEAKAVAYINVDVAVSGDTFTARTVPGL
KKVIQRAFDVANEEDEMKAANIITDDFDYTSDLTSFLTFAGIPVVNLAFERNEENPTPMP
FLGSCEDTVSWIDTFGSEYWENAARLGKIWSYLILFLANDPVVPYDLEDEINGVGEMLKR
IPEIPGANALDLRKINEEFSELLESLIRFEDEIREWKSLMMHNSYTVSVKKHPELEGYNA
KLARFERSFLDEAGLPGHEWYKHLIYGPNLRNSHSQLFPSIFDALLYGDVEAAQKEVKRI
ALALDRAHNEIRFA

As a zinc metalloprotease, it likely contains the characteristic HEXXH motif that coordinates the catalytic zinc ion, though detailed structural analysis would be required to confirm specific domain organization.

What expression systems have been successful for producing recombinant SPBC354.09c?

Recombinant full-length SPBC354.09c has been successfully expressed in E. coli with an N-terminal His tag . When planning expression experiments, researchers should consider:

• Using bacterial expression systems optimized for eukaryotic proteins

• Testing reduced expression temperatures (16-25°C) to improve proper folding

• Supplementing with zinc in the growth media to ensure proper metalloprotease formation

• Optimizing induction conditions to balance yield and solubility

What is the recommended protocol for transforming S. pombe for SPBC354.09c studies?

Based on established S. pombe transformation protocols, the following method is recommended:

This protocol has been optimized to minimize recombination between plasmids by sequential transformation .

What purification strategies are optimal for recombinant SPBC354.09c?

For His-tagged recombinant SPBC354.09c , a multi-step purification approach is recommended:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA or similar resin

• Additional purification by size-exclusion chromatography or ion exchange

• Buffer optimization to include zinc ions (typically 10-100 μM ZnCl2)

• Consideration of protease inhibitors to prevent auto-proteolysis

Proper storage conditions include avoiding repeated freeze-thaw cycles, with recommendations to store working aliquots at 4°C for up to one week and long-term storage at -20°C .

How can I design gene deletion experiments to study SPBC354.09c function?

Since SPBC354.09c is viable when deleted , gene deletion studies provide valuable insights into its function. Consider the following methodology:

• Create deletion cassettes with appropriate selection markers

• Transform S. pombe using the lithium acetate method described above

• Confirm deletion by PCR validation (average efficiency of gene deletion in S. pombe is approximately 51%)

• Test phenotypes under various conditions beyond standard growth

• Compare with orthologous gene deletion in S. cerevisiae (YPL176c) for evolutionary insights

What approaches can identify the specific proteolytic substrates of SPBC354.09c?

As an uncharacterized zinc metalloprotease, determining the natural substrates of SPBC354.09c requires sophisticated approaches:

• Comparative proteomics: Compare the proteome profiles of wild-type and SPBC354.09c deletion strains to identify proteins with altered abundance or processing

• TAILS (Terminal Amine Isotopic Labeling of Substrates): Enrich and identify N-terminal peptides generated by SPBC354.09c activity

• Peptide library screening: Test recombinant SPBC354.09c activity against diverse peptide substrates to establish cleavage site preferences

• Protein-protein interaction studies: Identify binding partners that may represent substrates using affinity purification coupled with mass spectrometry

How can I investigate the potential role of SPBC354.09c in stress response pathways?

While SPBC354.09c deletion strains are viable under standard conditions , metalloproteases often function in stress response pathways. To investigate this:

• Subject deletion strains to various stressors (oxidative, heat, osmotic, metal ions, cell wall stress)

• Analyze growth, survival, and morphological changes under stress conditions

• Perform transcriptomics comparing wild-type and deletion strains under stress

• Conduct genetic interaction screening to identify synthetic interactions with known stress response genes

• Analyze potential post-translational modifications of SPBC354.09c under stress conditions

What structure-function relationship studies would advance understanding of SPBC354.09c?

For detailed structure-function analysis:

• Site-directed mutagenesis: Target the predicted catalytic HEXXH motif and substrate-binding residues

• Domain mapping: Create truncation mutants to identify minimal functional domains

• Structural biology: Attempt crystallization of the recombinant protein or use cryo-EM

• Molecular dynamics simulations: Model substrate binding and catalytic mechanism

• Comparative analysis: Use structural information from better-characterized zinc metalloproteases to inform hypotheses

What are common challenges in working with recombinant SPBC354.09c?

Researchers should anticipate and address these potential issues:

How can I validate that my recombinant SPBC354.09c is properly folded and active?

To confirm proper folding and activity:

• Biophysical characterization: Circular dichroism to assess secondary structure; thermal shift assays for stability

• Activity assays: Test against generic metalloprotease substrates

• Metal content analysis: ICP-MS to confirm zinc incorporation at expected stoichiometry

• Controls: Compare activity to catalytically inactive mutants (e.g., HEXXH → AAXXA)

• Responsiveness to inhibitors: Test sensitivity to metalloprotease inhibitors (e.g., EDTA, 1,10-phenanthroline)

What are the key considerations for experimental reproducibility when working with SPBC354.09c?

To ensure reproducible results:

• Maintain consistent expression and purification protocols

• Document batch-to-batch variability in specific activity

• Include appropriate positive and negative controls in all experiments

• Standardize buffer components, especially metal ion concentrations

• Store protein as recommended (working aliquots at 4°C for up to one week, avoid repeated freeze-thaw cycles)

How should I interpret phenotypic data from SPBC354.09c deletion strains?

When analyzing phenotypic data:

• Compare growth rates and morphology under multiple conditions, not just standard media

• Consider that non-essential genes like SPBC354.09c often show condition-specific phenotypes

• Look for subtle phenotypes that may indicate regulatory rather than essential functions

• Analyze the effects of overexpression alongside deletion

• Consider genetic background effects by testing the deletion in multiple strain backgrounds

What statistical approaches are appropriate for analyzing SPBC354.09c enzymatic activity?

For rigorous enzymatic analysis:

• Determine standard enzyme kinetic parameters (Km, kcat, Vmax) using appropriate models

• Use multiple substrate concentrations to generate Michaelis-Menten or Lineweaver-Burk plots

• Perform triplicate measurements with appropriate controls

• Apply two-way ANOVA to compare activity under different conditions

• Use non-linear regression for inhibition studies and complex kinetic behaviors

This comprehensive FAQ collection should serve as a valuable resource for researchers at various stages of investigation into SPBC354.09c, from basic characterization to advanced functional studies.