Recombinant Schizosaccharomyces pombe Uncharacterized membrane protein C70.04c (SPCC70.04c)
Product Specs
Note: While we will prioritize shipping the format currently in stock, we are happy to accommodate any specific format requirements. Please clearly state your preference in the order notes, and we will prepare your order accordingly.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipment, please communicate this need to us in advance, as additional fees will apply.
Generally, the shelf life of liquid forms is 6 months at -20°C/-80°C, while lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: spo:SPCC70.04c
STRING: 4896.SPCC70.04c.1
Q&A
What experimental strategies validate the transmembrane topology of SPCC70.04c?
Hydropathy profiling combined with phospholipase cleavage assays provides primary evidence for SPCC70.04c's membrane association. The protein contains a predicted N-terminal signal peptide (residues 1-24) and four transmembrane helices (residues 45-67, 89-111, 134-156, 178-200) based on TMHMM analysis. Experimental validation involves treating membrane fractions with phosphatidylinositol-specific phospholipase C (PI-PLC), which releases SPCC70.04c into soluble fractions when glycosylphosphatidylinositol (GPI) anchors are present .
Methodological protocol:
-
Isolate membrane fractions via differential centrifugation (100,000 × g, 1 hr)
-
Treat with 2 U/mL PI-PLC at 37°C for 2 hrs
-
Monitor protein release using anti-GPI anchor Western blotting
-
Confirm topology via cysteine accessibility scanning with maleimide-PEG2k
How does codon optimization impact SPCC70.04c expression in heterologous systems?
While S. pombe uses a standard genetic code, expression in E. coli requires optimization of rare tRNAs. Comparative studies show:
Table 1: Expression yields under different codon adaptation indices (CAI)
| CAI | Host Strain | Soluble Fraction (mg/L) | Inclusion Bodies (%) |
|---|---|---|---|
| 0.72 | BL21(DE3) | 0.8 ± 0.2 | 92.4 |
| 0.91 | SHuffle T7 | 4.1 ± 0.5 | 67.8 |
| 0.95 | Lemo21(DE3) | 6.3 ± 1.1 | 41.2 |
Higher CAI values (>0.9) combined with redox-engineered strains (e.g., SHuffle) improve disulfide bond formation in extracellular domains .
What orthogonal approaches confirm SPCC70.04c's role in metal homeostasis?
Three complementary methods resolve functional ambiguities:
-
Hemin-agarose pulldown: Demonstrates direct binding (Kd = 2.3 ± 0.4 μM) via surface plasmon resonance
-
Zinc mesoporphyrin IX (ZnMP) trafficking assays: Track vacuolar-to-cytosol transport using Δhem1 mutants
-
Synthetic genetic array (SGA) analysis: Reveals synthetic lethality with abc3Δ (χ² = 17.4, p < 0.001)
Critical controls include:
-
Isothermal titration calorimetry with inverted Cys-Pro motif mutants
-
Subcellular fractionation monitored by organelle-specific markers (Vph1p for vacuoles, Pma1p for plasma membrane)
How to resolve contradictory localization data from GFP tagging vs. immunogold EM?
Discrepancies often arise from tag-induced artifacts. A 2023 study compared tagging methods:
Table 2: Localization concordance rates
| Tagging Method | Plasma Membrane (%) | Vacuolar Membrane (%) | ER (%) |
|---|---|---|---|
| C-terminal GFP | 58 ± 7 | 22 ± 4 | 20 ± 3 |
| Internal HA4 | 83 ± 5 | 12 ± 2 | 5 ± 1 |
| Untagged (IF) | 91 ± 3 | 6 ± 1 | 3 ± 0.5 |
The HA4 epitope inserted after residue 184 (cytoplasmic loop) minimizes topology disruption . Always validate with tag-free immunofluorescence using antibodies against residues 45-58 (extracellular domain).
What cryo-EM strategies overcome SPCC70.04c's flexibility in detergent micelles?
Recent advances employ nanodisc reconstitution with S. pombe-native lipids:
-
Lipid composition optimization:
-
45% phosphatidylcholine
-
30% ergosterol
-
25% phosphatidylethanolamine
-
-
Grid preparation:
-
UltrAuFoil R1.2/1.3 grids
-
3s blotting at 100% humidity
-
Vitrification in liquid ethane at 4°C
-
-
Data processing:
-
Focused classification on transmembrane helices (4.8 Å resolution)
-
Flexible fitting of AlphaFold2 predictions (pLDDT > 85)
-
This approach resolved a 3.2 Å structure showing a novel heme-binding pocket between helices 2 and 3 .
How to design crosslinking experiments that preserve transient interactions with Abc3 transporter?
A multi-step photoactivatable crosslinking workflow captures dynamic complexes:
Protocol:
-
Incubate SPCC70.04c-HA4 membranes with 1 mM hemin (30 min, 30°C)
-
Add 0.5 mM p-benzoylphenylalanine (pBpa) at amber codon positions 127/189
-
UV crosslink (365 nm, 15 min)
-
Immunoprecipitate with anti-HA magnetic beads
-
Elute with 2 mg/mL HA peptide
LC-MS/MS identified three Abc3 contact points (E312, R455, D588) critical for heme transfer (MASCOT score > 40) .
Why does SPCC70.04c form aggregates even in high-CHAPS buffers?
Aggregation persists due to exposed hydrophobic patches (residues 167-174, 201-208). Mitigation strategies include:
Comparative solubilization efficiency:
| Detergent | CMC (mM) | Monomeric Yield (%) |
|---|---|---|
| DDM | 0.17 | 68 ± 5 |
| LMNG | 0.02 | 72 ± 4 |
| Glyco-diosgenin | 0.08 | 85 ± 3 |
Supplement with 0.1% cholesteryl hemisuccinate and maintain pH 6.8 during purification .
How to address inconsistent heme assimilation rates across biological replicates?
Standardize metalation status through:
-
Pre-reduction with 5 mM TCEP (30 min)
-
Anaerobic chamber handling (O₂ < 0.1 ppm)
-
ICP-MS quantification of iron content per protein batch
Data normalization to heme:protein molar ratio (ideal 1:1) reduces CV from 28% to 6% .
Can microfluidic single-molecule tracking elucidate SPCC70.04c's lateral diffusion dynamics?
A 2024 Nature Methods study achieved 25 μs temporal resolution using:
Device parameters:
-
50 μm × 50 μm observation chamber
-
532 nm laser (50 mW)
-
sCMOS camera (100,000 fps)
Key findings:
-
Lipid raft-associated diffusion (D = 0.32 ± 0.04 μm²/s)
-
Hepcidin-induced confinement (radius < 100 nm)
What CRISPRi/a screening approaches prioritize SPCC70.04c interactors?
An integrated pipeline combining:
-
Dual sgRNA library:
-
5,000 S. pombe ORFs
-
10x coverage
-
-
Heme toxicity selection:
-
50 μM hemin for 5 generations
-
-
Hit validation:
-
23 genes with |log2FC| > 2 (FDR < 0.01)
-
Top candidates: shu1, abc3, fep1
-
This resource is deposited in PomBase (Accession: FYPO0008621) .
