Recombinant Shigella flexneri serotype 5b Cobalamin synthase (cobS)

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Cat. No.

BT2056756

Source

in vitro E.coli expression system

Synonyms

cobS; SFV_2064; Adenosylcobinamide-GDP ribazoletransferase; Cobalamin synthase; Cobalamin-5'-phosphate synthase

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Product Specs

Form

Lyophilized powder
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Lead Time

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Notes

Repeated freezing and thawing is not recommended. For optimal use, store working aliquots at 4°C for up to one week.

Reconstitution

We recommend centrifuging the vial briefly prior to opening to ensure the contents settle at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our default final concentration of glycerol is 50%. Customers can use this as a reference.

Shelf Life

Shelf life is influenced by various factors, including storage conditions, buffer ingredients, temperature, and the inherent stability of the protein itself.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.

Storage Condition

Upon receipt, store at -20°C/-80°C. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.

Tag Info

Tag type is determined during the manufacturing process.

The tag type is established during production. If you require a specific tag type, please inform us, and we will prioritize developing the specified tag.

Synonyms

cobS; SFV_2064; Adenosylcobinamide-GDP ribazoletransferase; Cobalamin synthase; Cobalamin-5'-phosphate synthase

Buffer Before Lyophilization

Tris/PBS-based buffer, 6% Trehalose.

Datasheet

Please contact us to get it.

Expression Region

1-247

Protein Length

full length protein

Species

Shigella flexneri serotype 5b (strain 8401)

Target Names

cobS

Target Protein Sequence

MSKLFWAMLSFITRLPVPRRWSQGLDFEHYSRGIITFPLIGLLLGAISGLVFMVLQAWCG APLAALFSVLVLALMTGEFHLDGLADTCDGVFSARSRDRMLEIMRDSRLGTHGGLALIFV VLAKILVLSELALRGEPILASLAAACAVSRGTAALLMYRHRYAREEGLGNVFIGKIDGRQ TCVTLGLAAIFAAVLLLGMHGVAAMVVTMVAIFILGQLLKRTLDGQTGDTLGAAIELGEL VFLLALL

Uniprot No.

Q0T3C3

Target Background

Function

Catalyzes the synthesis of adenosylcobalamin (Ado-cobalamin) by joining adenosylcobinamide-GDP and alpha-ribazole. It also synthesizes adenosylcobalamin 5'-phosphate from adenosylcobinamide-GDP and alpha-ribazole 5'-phosphate.

Database Links

KEGG: sfv:SFV_2064

Protein Families

CobS family

Subcellular Location

Cell inner membrane; Multi-pass membrane protein.

Q&A

Basic Research Questions

Q1: What are optimal conditions for expressing recombinant cobS in E. coli?

Recombinant cobS expression in E. coli requires:

Vector selection: Use pET-derived plasmids with T7 RNA polymerase-dependent promoters .
Solubility tags: N-terminal His-tag enhances purification efficiency (SDS-PAGE >90% purity) .
Induction parameters:
- Optimal induction at 16–25°C with 0.1–1 mM IPTG for 16–20 hours to minimize inclusion body formation .
Strain choice: BL21(DE3) or derivatives for high yield and proper disulfide bond formation .

Q2: How is cobS purified from E. coli lysates?

Step	Method	Rationale
1. Cell lysis	Sonication or enzymatic lysis (e.g., lysozyme)	Preserves membrane-bound regions if retained .
2. Clarification	Centrifugation (20,000 × g, 30 min)	Removes insoluble debris .
3. Affinity chromatography	Ni-NTA resin	Selective binding to His-tag .
4. Buffer exchange	Dialysis or gel filtration	Removes imidazole, stabilizes protein in Tris/PBS buffer with 6% trehalose .

Q3: What primary functional assays validate cobS activity?

Key assays include:

Substrate binding analysis: Use radiolabeled GDP ribazole or adenosylcobinamide phosphate to measure binding affinity (Kd) .
Enzymatic activity: Monitor conversion of adenosylcobinamide phosphate to adenosylcobalamin using HPLC or LC-MS .
Membrane association: Confirm via alkaline phosphatase treatment of membrane fractions or liposome reconstitution .

Advanced Research Questions

Q4: How do environmental factors influence cobS function in Shigella pathogenesis?

Factor	Impact on cobS	Experimental Model
Bile salts	Induce biofilm formation and OMV production	In vitro small intestine-like conditions (TSB + bile salts)
Glucose	Enhances adherence gene expression	Human intestinal organoid-derived epithelial monolayers (HIODEMs)
Host cell interaction	Potential packaging into OMVs	Proteomic analysis of OMV content post-infection

Q5: What structural insights inform cobS biochemical studies?

Membrane integration:
- CobS is a polytopic inner membrane protein; reconstitute purified protein into liposomes to study lipid bilayer effects .
- Use protease protection assays to confirm topology .
Residue-specific analysis:
- Mutational studies targeting conserved motifs (e.g., Walker A/B motifs) to identify catalytic residues .
- Densitometry quantification of purified protein homogeneity (96% purity) .

Q6: How does cobS relate to Shigella virulence plasmid-encoded factors?

Factor	Role	cobS Interaction
PhoP-PhoQ system	Regulates OMV production and outer membrane protein expression	May modulate cobS packaging into OMVs
T3SS effectors	Facilitate invasion and host cell manipulation	Potential cross-regulation in stress response pathways

Data Contradiction Analysis

Q7: Why do reports conflict about cobS solubility in recombinant systems?

Observation	Explanation
Membrane-bound in S. Typhimurium	Native localization requires lipid environment; recombinant expression may yield soluble forms due to tag placement or truncation .
Soluble in E. coli lysates	Full-length cobS with His-tag may adopt soluble conformation in E. coli cytoplasm; requires reconstitution for membrane activity .

Q8: How to resolve discrepancies in cobS OMV association?

Experimental variables:
- Growth media: TSB vs. LB affects OMV biogenesis .
- Virulence plasmid presence: Deletion mutants show reduced OMV production .
Validation methods:
- Proteomic profiling of OMV fractions using LC-MS/MS .
- Confocal microscopy with anti-cobS antibodies in infected cells .

Comparative Genomics and Evolutionary Analysis

Q9: What distinguishes S. flexneri cobS from other Shigella/Enterobacteria?

Species	Key Feature
S. flexneri	Full-length sequence (247 aa) with His-tag compatibility; expressed in E. coli for structural studies .
S. Typhimurium	96% homogeneous protein post-purification; membrane association critical for activity .
E. coli	Lacks cobS homologs; relies on alternative cobalamin salvage pathways.

Q10: How to design homology-based mutagenesis for cobS?

Sequence alignment: Identify conserved motifs (e.g., Walker A/B) across Shigella spp. and archaeal homologs .
Site-directed mutagenesis: Target residues in substrate-binding pockets or transmembrane domains .
Functional screening: Use high-throughput assays (e.g., colorimetric GDP ribazole transferase activity) to prioritize variants .

Methodological Recommendations

Q11: What controls are essential for cobS functional studies?

Control Type	Purpose
Wild-type E. coli lysate	Baseline activity in non-recombinant systems .
Heat-denatured cobS	Negative control for enzymatic activity assays .
Liposome-only samples	Exclude substrate binding to lipids alone .

Q12: How to assess cobS stability under storage conditions?

Parameter	Method
Thermal stability	Circular dichroism (CD) spectroscopy at 25–95°C .
Freeze-thaw cycles	SDS-PAGE analysis post-thaw to detect aggregation .
Prolonged storage	Activity retention assays after 6 months at -80°C .

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Recombinant Shigella flexneri serotype 5b Cobalamin synthase (cobS)

Product Specs

Target Background

Q&A

Basic Research Questions

Q1: What are optimal conditions for expressing recombinant cobS in E. coli?

Q2: How is cobS purified from E. coli lysates?

Q3: What primary functional assays validate cobS activity?

Advanced Research Questions

Q4: How do environmental factors influence cobS function in Shigella pathogenesis?

Q5: What structural insights inform cobS biochemical studies?

Q6: How does cobS relate to Shigella virulence plasmid-encoded factors?

Data Contradiction Analysis

Q7: Why do reports conflict about cobS solubility in recombinant systems?

Q8: How to resolve discrepancies in cobS OMV association?

Comparative Genomics and Evolutionary Analysis

Q9: What distinguishes S. flexneri cobS from other Shigella/Enterobacteria?

Q10: How to design homology-based mutagenesis for cobS?

Methodological Recommendations

Q11: What controls are essential for cobS functional studies?

Q12: How to assess cobS stability under storage conditions?

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