Recombinant Human CA4 Protein: Properties & Research Uses

Recombinant Human CA4 Protein is a GPI-anchored, membrane-associated carbonic anhydrase isoform that catalyzes the rapid interconversion of carbon dioxide and bicarbonate, making it a critical tool for studying acid-base physiology, renal function, and respiratory CO2 transport in vitro.

Protein Name

Recombinant Human Carbonic Anhydrase 4 (CA4)

Anchoring Mechanism

GPI (glycosylphosphatidylinositol) anchor — membrane-bound, extracellular-facing

Primary Biological Function

Catalyzes reversible hydration of CO2 to bicarbonate and proton (CO2 + H2O ⇌ HCO3⁻ + H⁺)

Primary Expression Sites

Kidney proximal tubule, lung capillaries, heart, skeletal muscle

Gene / UniProt

CA4 gene; UniProt accession P22748 (Human)

Recommended Applications

Enzyme activity assays, protein interaction studies, Western blot, ELISA, functional rescue experiments

What Is Carbonic Anhydrase 4 (CA4)?

Carbonic anhydrase 4 (CA4) is a member of the α-carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide: CO₂ + H₂O ⇌ HCO₃⁻ + H⁺. Among the 15 human CA isoforms, CA4 is distinctive because it is anchored to the extracellular face of the plasma membrane via a glycosylphosphatidylinositol (GPI) lipid anchor, placing its active site in direct contact with the extracellular milieu.

The CA4 gene encodes a ~35 kDa mature polypeptide. The protein contains a catalytic zinc ion coordinated by three conserved histidine residues, a structural arrangement shared across the α-CA family. CA4 is highly expressed in the kidney proximal tubule, lung capillary endothelium, heart, and skeletal muscle, where rapid CO₂/bicarbonate equilibration is physiologically essential for acid-base homeostasis and gas exchange.

Mutations or loss of CA4 activity have been linked to retinitis pigmentosa type 17 (RP17), underscoring the enzyme's importance beyond classical acid-base physiology and making it an active target in ocular disease research.

Biochemical Properties of Recombinant Human CA4 Protein

Recombinant Human CA4 Protein is produced using heterologous expression systems to yield a soluble or appropriately processed form of the native GPI-anchored enzyme. The recombinant form retains the core catalytic domain and zinc-binding site, enabling authentic enzymatic activity measurements in solution-phase assays.

Key biochemical characteristics include:

• Catalytic mechanism: Zinc-hydroxide mechanism common to α-carbonic anhydrases, with the zinc-bound hydroxide acting as the nucleophile for CO₂ hydration.
• Active site: Contains a catalytic Zn²⁺ ion coordinated by His94, His96, and His119 (human CA4 numbering, needs verification against primary literature).
• Post-translational modifications: The native protein is N-glycosylated and GPI-anchored; recombinant preparations may vary in glycosylation state depending on the expression host.
• Stability: Zinc metalloenzymes of this class are generally stable at 4 °C in physiological buffers; long-term storage typically requires –20 °C or –80 °C with a carrier protein such as BSA.

Researchers should confirm purity (typically ≥90% by SDS-PAGE), endotoxin levels, and specific activity (units/mg) from the certificate of analysis (CoA) supplied with each lot.

Research Applications

Recombinant Human CA4 Protein supports a broad range of in vitro research applications:

1. Enzyme activity assays: The protein serves as a positive control or active enzyme source in colorimetric or stopped-flow CO₂ hydration assays, enabling kinetic characterization (Km, Vmax, kcat) and inhibitor screening (e.g., sulfonamide-class carbonic anhydrase inhibitors).
2. Inhibitor/drug discovery: CA4 is a validated target for isoform-selective carbonic anhydrase inhibitor development. Recombinant CA4 enables fluorescence thermal shift assays, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) binding studies.
3. Western blot and ELISA: As an antigen standard for antibody validation or quantitative immunoassay development, recombinant CA4 provides a well-defined reference protein.
4. Protein–protein interaction studies: Pull-down or co-immunoprecipitation experiments can employ recombinant CA4 to map interaction partners in carbonic anhydrase-associated signaling or transport complexes.
5. Disease model research: Given CA4's association with RP17 and renal acidification, recombinant protein supports mechanistic studies exploring pathological variants and rescue of enzyme function.

Always verify that the expression host and tag configuration are compatible with your specific assay format before use.

Handling, Storage, and Quality Considerations

Proper handling of Recombinant Human CA4 Protein is essential to maintain enzymatic activity and experimental reproducibility:

• Storage temperature: Store at –20 °C for short-term use; –80 °C is recommended for long-term storage. Avoid repeated freeze-thaw cycles by preparing single-use aliquots upon first thaw.
• Reconstitution: If supplied lyophilized, reconstitute in the buffer recommended in the product datasheet (typically sterile PBS or Tris-HCl with a stabilizer such as glycerol or BSA). Allow the vial to equilibrate at room temperature before opening.
• Buffer compatibility: Avoid buffers containing strong reducing agents (e.g., high-concentration DTT or β-mercaptoethanol) that may affect zinc coordination, unless validated for your specific preparation.
• Endotoxin: For cell-based assays, confirm endotoxin content (EU/mg) from the CoA. Select low-endotoxin preparations when working with primary cells or sensitive in vitro models.
• Activity verification: Upon receipt, it is best practice to verify enzymatic activity using a validated assay before committing large volumes to experiments.

Refer to the Aladdin Scientific product datasheet for lot-specific purity, concentration, formulation, and activity data for SKU rp365577.

Frequently asked questions

What expression system is used to produce Recombinant Human CA4 Protein (rp365577)?

The specific expression system (e.g., E. coli, HEK293, baculovirus/insect cells) for rp365577 should be confirmed in the Aladdin Scientific product datasheet or certificate of analysis, as this affects glycosylation state and activity. Please contact Aladdin Scientific technical support or download the CoA for lot-specific details.

Can Recombinant Human CA4 Protein be used for inhibitor screening assays?

Yes. Recombinant Human CA4 Protein is suitable for enzyme inhibition assays targeting the carbonic anhydrase active site. Common formats include stopped-flow CO₂ hydration assays, fluorescence-based assays, and biophysical binding assays (SPR, ITC, thermal shift). Researchers should verify that the recombinant preparation retains catalytic zinc and measurable specific activity before use in high-throughput screens.

How should I store Recombinant Human CA4 Protein to preserve its activity?

Store Recombinant Human CA4 Protein at –80 °C for long-term preservation. Prepare single-use aliquots upon first thaw to avoid repeated freeze-thaw cycles, which can cause protein aggregation and loss of enzymatic activity. If supplied lyophilized, reconstitute per the datasheet instructions and store aliquots accordingly.

Is Recombinant Human CA4 Protein suitable for use as a Western blot molecular weight standard or antigen?

Recombinant Human CA4 Protein can serve as a positive control antigen in Western blot experiments for anti-CA4 antibody validation, provided purity is ≥90% as confirmed by SDS-PAGE. It can also be used to generate standard curves in quantitative ELISA formats. Note that glycosylation differences between the recombinant and endogenous forms may lead to slightly different apparent molecular weights on SDS-PAGE.

References

1. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem. 1995;64:375-401. doi:10.1146/annurev.bi.64.070195.002111
2. Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008;7(2):168-181. doi:10.1038/nrd2467
3. Stams T, Christianson DW. X-ray crystallographic studies of mammalian carbonic anhydrase isozymes. EXS. 2000;90:159-174. PMID:10986446
4. Rebello G, et al. Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa. Proc Natl Acad Sci USA. 2004;101(18):6987-6992. doi:10.1073/pnas.0401529101

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.