| Field | Specification |
|---|---|
| Mfr No | |
| Activity | |
| Conjugate | |
| Endotoxin Level | |
| Expression System | |
| Form | Liquid or Lyophilized powder |
| Molecular Weight | |
| Product Type | |
| Protein Length | |
| Purity | |
| Reconstitution | |
| Species | |
| Storage | |
| Target | |
| UniProt # |
Overview
Recombinant Clostridioides difficile Toxin B (toxB), partial is a recombinant protein preparation from Clostridioides difficile (Peptoclostridium difficile) designed for use in assay development, binding studies, and functional characterization. Key attributes such as expression system, expressed region, and affinity tag(s) help researchers match the reagent to specific experimental readouts.
Key elements and design rationale
- Expression system: E.coli expression is commonly used for rapid, scalable production. For targets that require glycosylation or other post-translational modifications, consider how a prokaryotic system may affect folding or activity.
- Expression region: The expressed fragment (2-543aa) focuses the reagent on a defined domain/segment, which can influence binding interfaces and epitope availability.
- Tag(s)/format: His/Myc tags can support purification and detection in pull-down or binding assays; confirm that the tag position does not interfere with the interaction of interest.
- Purity: ≥85% (SDS-PAGE) provides a quick checkpoint for reagent quality in downstream analytical workflows.
- Form: Supplied as Liquid or Lyophilized powder; select the format that best fits your lab’s handling and aliquoting preferences.
Recombinant design choices (expression host, fragment boundaries, and tag configuration) help balance yield, solubility, and assay compatibility. Choose conditions and controls that match the recombinant format to your experimental question.
Biological background
toxB has been reported to be involved in Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis. TcdB constitutes the main toxin that mediates the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted. Compared to TcdA, TcdB is more virulent and more important for inducing the host inflammatory and innate immune responses. This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdB) into the host cytosol. Targets colonic epithelia by binding to the frizzled receptors FZD1, FZD2 and FZD7, and enters host cells via clathrin-mediated endocytosis. Frizzled receptors constitute the major host receptors in the colonic epithelium, but other receptors, such as CSPG4 or NECTIN3/PVRL3, have been identified. Binding to carbohydrates and sulfated glycosaminoglycans on host cell surface also contribute to entry into cells. Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane. This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the cytosol. ; [Glucosyltransferase TcdB]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases. Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, RhoG and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor. Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function.. When interpreting results, consider species context, domain architecture, and whether the recombinant format represents full-length or a defined region.
Research relevance and current trends
- Antigen and virulence-factor studies that compare strain- or domain-specific binding and immune recognition.
- Use of recombinant proteins as standards for quantitative assays and serology-oriented method development.
Common research applications
- Binding and interaction assays: quantify partner binding and rank conditions using plate-based formats or biophysical methods (SPR/BLI).
- Enzymology: assess catalytic activity and compare substrate preferences or inhibitor effects using appropriate controls.
- Assay development: use as a standard, spike-in control, or positive control where consistent specifications are required.
Interpretation typically relies on relative comparisons (treated vs control, mutant vs wild-type, or dose/time series) using consistent sample handling and appropriate normalization.
Notes for experimental interpretation
- Post-translational modifications: expression system can affect glycosylation and processing; interpret differences cautiously when comparing to native protein.
- Isoforms and domains: expressed regions may not capture all isoform-specific features; match fragment boundaries to your assay’s binding site.
- Controls: include blank matrix controls, tag-only controls (where relevant), and orthogonal readouts (e.g., WB/qPCR/ELISA) to support interpretation.
What is protein expression and purification?
Why is there no/low protein expression?
b. Rare codons. You should optimize codons, use strains supplementing rare codons, induce at lower temperature or grow in poor media.
c. Protein toxicity. You should use promoters with tighter regulation or lower plasmid copy number. Use pLysS/pLysE bearing strains in T7-based systems or strains that are better for the expression of toxic proteins. Start induction at high OD and shorten induction time. Add glucose when using expression vectors containing lac-based promoters.
How to avoid inclusion bodies and improve soluble expression?
b. Incorrect disulfide bond formation. You should add fusion partners, including thioredoxin, DsbA, DsbC. Clone in a vector containing secretion signal peptide to cell periplasm. Use gamiB (DE3)strains with oxidative cytoplasmic environment. Lower inducer concentration and induction temperature.
c. Incorrect folding. You should use a fusion partner. Co-express with molecular chaperones. Use strains with cold-adapted chaperones. Supplement media with chemical chaperones and cofactors. Reduce the inducer concentration and add fresh media. Induce for a shorter time at low temperature.
Why is the molecular weight of protein smaller than the predicted?
b. Imbalanced translation process of fusion protein. You should change another fusion tag or move fusion tag to C-terminal. You should induce for a shorter time at low temperature or change to poor media.
c. Protein degradation. You should replace specific protease sites. Use protease deficient strains. Induce at high OD. You should induce for a shorter time at low temperature or use protease inhibitors when breaking cells.
Why is the actual band size different from the predicted?
b. Post-translational cleavage. Many proteins are synthesized as pro-proteins, and then cleaved to give the active form.
c. Splice variants. Alternative splicing may create different sized proteins from the same gene.
d. Relative charge. The composition of amino acids have different relative charge which will affect the electrophoretic mobility.
e. Multimers such as dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands.
f. Protein structure such as disulfide bond, protein secondary structure or protein 3D structure formation.
g. Hydrophobic proteins, such as transmembrane proteins, may have difficulties in migrating into the gel, and thus resulting in different multi-banded patterns.
How to express a protein with bioactivity? Why is the protein inactive?
a. Low solubility of the protein. You should fuse desired protein to a fusion partners and lower temperature.
b. Lack of essential post translational modification. You should change another expression system.
c. Incomplete folding. You should use a fusion partner and use strains with cold-adapted chaperones. Co-express with molecular chaperones at lower temperature. Monitor disulfide bond formation and allow further folding in vitro.
d. Mutations in cDNA. You should sequence plasmid before and after induction or use a recA− strain to ensure plasmid stability. Transform E. coli before each expression round.
Why are our protein products almost invisible in pipes?
Tips: Before opening the lid, we recommend to centrifuge in a small centrifuge for 20-30 seconds firstly to ensure that the contents are on the bottom of the tube. Our quality control steps ensure that the amount of protein contained in each tube is accurate, although sometimes you can’t see the protein powder, but the protein content in the tube is still very accurate.
How is the protein purified? Is the purity guaranteed?
Although we guarantee a minimum purity standard of >85%, some of the proteins we prepared have a purity of 95% or even 97%.
How should I reconstitute and store the products?
As for short-term storage or usage, please use sterile deionized water to completely reconstitute proteins to 0.1-1.0 mg/mL. Aliquot after 10-15 minutes if needed and store at 4℃.
As for long-term storage, the cytokines or recombinant proteins are recommended to add 5-50% of glycerol (final concentration) and aliquot for long-term storage at -20℃/-80℃. Our default final concentration of glycerol is 50%. Customers could use it as reference.
What types of tags do you use for fusion?
What is the impact of a given tag type and any potential biological activity of the protein?
Can you remove the endotoxin?
Can you offer aseptic manufacture processing?
How to determine species cross-reactivity of cytokines?
b. Many mouse cytokines may also have effect on human cells, however, the activity may be lower than the corresponding human cytokines.
c. One of the few human cytokines will be more active than corresponding mouse cytokines when acting on mouse cells, such as IL-7.
d. Interferon, GM-CSF, IL-3 and IL-4 and other cytokines are species-specific and almost have no activity on non-homologous cells.
e. In contrast, fibroblast growth factor (FGF) and neurotrophin are highly conserved and both have good activity on cells of different species.
What is the general preservative? Which kind of preservative do you usually add?
What is the general protectant? What kind of protectant do you usually add?
Can’t Find What You’re Looking For? We can help you source the best match or customize a recombinant protein solution for your study. Options may include species (human/mouse/rat), protein region/domain (full-length vs fragment), tag or label (His/GST/FLAG/biotin/fluorescent), expression system (E. coli/HEK293/insect), purity grade, formulation (buffer, carrier-free, glycerol-free), activity/functional validation (binding or enzymatic assays), endotoxin level (low-endotoxin for cell-based work), mutants/variants (point mutations, isoforms), and bulk or custom packaging. Click Talk to a Scientist to submit a request form, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
