Recombinant Capsular polysaccharide biosynthesis protein CapA (capA)

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Cat. No.

BT2454245

Source

in vitro E.coli expression system

Synonyms

capA; Capsular polysaccharide biosynthesis protein CapA

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Description

Definition and Biological Role

The recombinant Capsular Polysaccharide Biosynthesis Protein CapA (capA) is a critical component in bacterial capsular polysaccharide (CP) synthesis, particularly in Gram-positive pathogens like Staphylococcus aureus, Francisella tularensis, and Lactobacillus plantarum. It functions as a co-polymerase or kinase activator, regulating key enzymatic steps in CP precursor biosynthesis and polymerization .

Key Functions:

Tyrosine Kinase Activation: In S. aureus, CapA1 activates the CapB1 kinase, enabling phosphorylation of target enzymes (e.g., CapM, CapE) to modulate precursor synthesis .
Lipid Carrier Recycling: CapA1 cleaves pyrophosphate linkages in lipid-linked CP precursors (e.g., C55P), releasing undecaprenyl-phosphate for reuse in peptidoglycan biosynthesis .
Polysaccharide Assembly: CapA facilitates the attachment of CP precursors to murein (peptidoglycan) via interactions with LcpC-like transferases .

S. aureus Pathway:

CapA1B1 Complex Activation: Autophosphorylated CapB1 transfers phosphate to CapA1, enabling its dual role as a kinase activator and phosphodiesterase .
CapM Phosphorylation: Tyrosine phosphorylation of CapM by CapA1B1 increases lipid I cap synthesis by 4-fold, prioritizing CP precursor formation over peptidoglycan synthesis .
Lipid Precursor Processing: CapA1 hydrolyzes pyrophosphate bonds in lipid I/II cap, releasing C55P for reutilization .

F. tularensis Pathway:

Polysaccharide Assembly: CapA likely coordinates with CapB and CapC through its disordered domain to polymerize precursors, forming virulence-associated capsules .

Enhanced Exopolysaccharide Production

**Case Study: Lactobacillus plantarum P1**

Parameter	Wild-Type (P1)	P1-capA Overexpressed	Increase (%)
CPS Production	N/A	11.3 mg/L	N/A
EPS Thickness	0.0786 ± 0.0224 μm	0.1160 ± 0.0480 μm	48%
DBP Adsorption	N/A	2× Improvement	N/A

Overexpression of capA in L. plantarum doubled di-n-butyl phthalate (DBP) adsorption by thickening the exopolysaccharide layer .

Vaccine Development

Recombinant CapA from S. aureus (aa 1–221) is produced via E. coli, yeast, or baculovirus systems for vaccine research. It serves as a target antigen due to its conserved role in capsule biosynthesis and pathogenicity .

Research Findings and Data

Study Focus	Organism	Key Discovery	Methodology
CapA1 Kinase Role	S. aureus	CapA1B1-mediated phosphorylation of CapM enhances lipid I cap synthesis.	In vitro kinase assay
Structural Insights	F. tularensis	Disordered periplasmic domain critical for CapB/C interactions.	CD spectroscopy, DLS
Bioremediation	L. plantarum	capA overexpression increases EPS thickness and DBP adsorption capacity.	RT-qPCR, TEM imaging

Future Directions

Drug Targeting: CapA’s role in CP biosynthesis makes it a candidate for anti-virulence therapies in S. aureus and F. tularensis .
Industrial Applications: Engineering CapA in probiotics (e.g., L. plantarum) for enhanced bioremediation or biofilm formation .

Product Specs

Form

Lyophilized powder
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized preparation.

Lead Time

Delivery times vary depending on the purchase method and location. Please consult your local distributor for precise delivery estimates.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is requested in advance. Additional fees apply for dry ice shipping.

Notes

Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.

Reconstitution

Centrifuge the vial briefly before opening to consolidate the contents. Reconstitute the protein in sterile, deionized 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 standard glycerol concentration is 50%, provided as a guideline for your reference.

Shelf Life

Shelf life depends on several factors including storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life 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 determined during production. If a specific tag type is required, please inform us, and we will prioritize its development.

Synonyms

capA; Capsular polysaccharide biosynthesis protein CapA

Buffer Before Lyophilization

Tris/PBS-based buffer, 6% Trehalose.

Datasheet

Please contact us to get it.

Expression Region

1-221

Protein Length

full length protein

Species

Staphylococcus aureus

Target Names

capA

Target Protein Sequence

MESTIDLSELLGRVRKNMKLLIILPLLGLLISAIISFFFLDVKYQASTQILVNQKGNDSQ IMAQEVQSNIQLVNTYSEIVKSPRILDKVSKELDDKYSRSEISSMLTVTNQAESQVLNID VESKSGSNSEKIANKIAEVFSDEVPDIMNVDNVSVLSTADNTGKQVAPKPMVNLVVGLVI GLVIALLIIFIKEVFDKRIKTEEEVENELVIPVLGSIQKFD

Uniprot No.

P39850

Target Background

Function

Essential for the biosynthesis of type 1 capsular polysaccharide.

Protein Families

CpsC/CapA family

Subcellular Location

Cell membrane; Multi-pass membrane protein.

Q&A

Frequently Asked Questions on Recombinant Capsular Polysaccharide Biosynthesis Protein CapA (capA) in Academic Research

What experimental approaches are used to validate CapA expression and localization in bacterial systems?

Methodology:
- Cloning & Expression: Amplify capA via PCR using strain-specific primers (e.g., Cj0629 primers for Campylobacter jejuni) . Ligate into expression vectors (e.g., pCRT7/NT-TOPO, pMAL-C2x) .
- Immunoblotting: Use polyclonal antisera raised against recombinant CapA to detect ~116 kDa protein in cell lysates . Subcellular fractionation confirms outer membrane localization .
- Functional Inhibition: Treat cells with globomycin (signal peptidase II inhibitor) to block CapA export, confirming its lipoprotein nature .
Key Data:

Strain CapA Detection Rate Localization
C. jejuni NCTC11168 100% Outer membrane
Human fecal isolates 36% Variable

Strain	CapA Detection Rate	Localization
C. jejuni NCTC11168	100%	Outer membrane
Human fecal isolates	36%	Variable

How does CapA contribute to bacterial adhesion and pathogenesis?

Methodology:
- Mutagenesis: Generate capA insertion mutants via chloramphenicol/kanamycin cassette integration .
- In Vitro Assays: Compare wild-type and mutant strains in Caco-2 cell adhesion/invasion assays .
- In Vivo Models: Assess chicken colonization persistence over 14 days .
Findings:
- Adhesion: Mutants show 60–75% reduction in Caco-2 cell association .
- Colonization: Mutants fail to persist in chicken ceca beyond 7 days .

What are the optimal conditions for recombinant CapA expression in heterologous systems?

Methodology:
- Host Selection: Use Pichia pastoris for eukaryotic expression (e.g., Aspergillus niger CapA) .
- Activity Assays: Test enzymatic activity at pH 4.0–8.0 and 40–50°C .
Key Parameters:

Parameter Optimal Value Stability Range
pH 6.0 4.0–8.0 (60% retention)
Temperature 45°C 40–50°C (80% retention)
Metal Effects Mg²⁺ enhances; Cu²⁺/Fe²⁺ inhibit —

Parameter	Optimal Value	Stability Range
pH	6.0	4.0–8.0 (60% retention)
Temperature	45°C	40–50°C (80% retention)
Metal Effects	Mg²⁺ enhances; Cu²⁺/Fe²⁺ inhibit	—

How does tyrosine phosphorylation regulate CapA activity in Staphylococcus aureus?

Methodology:
- Kinase Assays: Incubate CapA1B1 complex with γ-³³P-ATP to detect autophosphorylation .
- Phosphosite Mapping: Use NetPhos 3.1 to predict Tyr157 as the critical regulatory site in CapM .
- Activity Modulation: Compare lipid I cap synthesis rates in phosphorylated vs. non-phosphorylated CapM .
Findings:
- Phosphorylation increases CapM glycosyltransferase activity by 4-fold .
- Tyr157 mutation (Y157F) abolishes activity enhancement .

What structural features enable CapA to interact with lipid-linked polysaccharide precursors?

Methodology:
- Domain Analysis: Predict transmembrane helices and hydrophilic regions via PSIPRED .
- Biophysical Studies: Use circular dichroism to assess α-helix/β-sheet content in CapA-frGFP fusions .
Key Insights:
- Membrane Anchoring: Two transmembrane domains (aa 1–22, 380–403) anchor CapA to the inner membrane .
- Substrate Binding: Hydrophilic domain (aa 50–300) contains disordered regions that adopt α-helical conformations under high ionic strength .

How can contradictions in CapA’s role across species be resolved?

Case Study:
- Bacillus anthracis: CapA is dispensable for capsule formation in Ames strain but critical in Pasteur II strain .
- Resolution Strategy:
  - Genetic Complementation: Express acpA/acpB in pag knockout mutants to restore capsule synthesis .
  - qPCR Profiling: Compare capABCD operon transcription levels in wild-type vs. mutant strains .
- Data:
  
  Strain capABCD Expression (vs. WT) Capsule Production
  Pasteur II Δpag 25% Absent
  Δpag + acpA 98% Restored

Strain	capABCD Expression (vs. WT)	Capsule Production
Pasteur II Δpag	25%	Absent
Δpag + acpA	98%	Restored

Methodological Guidance for Contradictory Data

Root-Cause Analysis Framework:
- Strain-Specific Factors: Compare genetic backgrounds (e.g., pXO1 plasmid presence in B. anthracis) .
- Environmental Conditions: Test capsule production under varying CO₂ levels or host-mimicking media .
- Functional Redundancy: Screen for paralogs (e.g., CapB in C. jejuni) using BLASTp and knockout assays .

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Recombinant Capsular polysaccharide biosynthesis protein CapA (capA)

Description

Definition and Biological Role

Enhanced Exopolysaccharide Production

**Case Study: Lactobacillus plantarum P1**

Vaccine Development

Research Findings and Data

Future Directions

Product Specs

Target Background

Q&A

Frequently Asked Questions on Recombinant Capsular Polysaccharide Biosynthesis Protein CapA (capA) in Academic Research

What experimental approaches are used to validate CapA expression and localization in bacterial systems?

How does CapA contribute to bacterial adhesion and pathogenesis?

What are the optimal conditions for recombinant CapA expression in heterologous systems?

How does tyrosine phosphorylation regulate CapA activity in Staphylococcus aureus?

What structural features enable CapA to interact with lipid-linked polysaccharide precursors?

How can contradictions in CapA’s role across species be resolved?

Methodological Guidance for Contradictory Data

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