Recombinant Human CENPE Protein: Research Guide & Protocol

Recombinant Human CENPE Protein is a key mitotic kinesin motor protein that plays an essential role in chromosome congression, spindle assembly checkpoint signaling, and kinetochore–microtubule interactions during cell division.

Protein Name

Centromere Protein E (CENPE)

Protein Family

Kinesin family, kinesin-7 subfamily

Primary Biological Function

Chromosome congression and spindle assembly checkpoint regulation during mitosis

Species Origin

Human (Homo sapiens)

Product SKU

rp371535

Typical Research Applications

Mitosis studies, kinetochore biology, biochemical assays, drug target validation

Biological Background of CENPE

Centromere Protein E (CENPE) is a large, plus-end-directed kinesin motor protein belonging to the kinesin-7 subfamily. In human cells, CENPE localizes to the outer kinetochore plate during mitosis, where it plays a pivotal role in chromosome congression — the process by which sister chromatid pairs align at the metaphase plate prior to segregation.

CENPE works in concert with the mitotic checkpoint complex (MCC) and interacts with checkpoint kinases such as BubR1 (BUB1B) to sustain spindle assembly checkpoint (SAC) signaling. Loss of functional CENPE leads to mitotic errors including chromosome misalignment, lagging chromosomes, and aneuploidy, which are hallmarks of genomic instability observed in various cancers. Consequently, CENPE has attracted attention as a potential anticancer drug target, with small-molecule inhibitors under investigation in oncology research.

Product Specifications & Biochemical Properties

Recombinant Human CENPE Protein (SKU: rp371535) is produced using recombinant expression systems to ensure consistency and reproducibility across experiments. Researchers should consult the accompanying Certificate of Analysis (CoA) for lot-specific data including purity, concentration, and formulation details, as these parameters may vary between production lots.

Key considerations for this recombinant protein include:

• Purity: Typically assessed by SDS-PAGE and/or HPLC; consult the CoA for lot-specific purity data.
• Formulation: Supplied in a stabilizing buffer; refer to the product datasheet for exact composition, as optimized formulations preserve motor domain activity.
• Storage: Generally recommended at -80 °C for long-term storage; avoid repeated freeze-thaw cycles to maintain biological activity.
• Biological Activity: Functional validation may include microtubule-binding assays or ATPase activity measurements; verify with the product datasheet.

Note: Specific numerical values for molecular weight, purity percentage, and buffer composition should be confirmed against the current product datasheet and CoA available from Aladdin Scientific.

Recommended Handling & Storage Protocol

Proper handling of Recombinant Human CENPE Protein is critical to preserve its structural integrity and biological activity. Follow the guidelines below to ensure optimal experimental outcomes:

1. Upon Receipt: Inspect the shipment immediately. Confirm the product has been maintained at the appropriate cold-chain temperature. Record the lot number and expiry date.
2. Reconstitution (if lyophilized): Allow the vial to equilibrate to room temperature for approximately 10–15 minutes before opening. Reconstitute with the volume of ultrapure water or the specified buffer indicated on the datasheet. Gently mix by pipetting; do not vortex, as vigorous agitation may denature the protein.
3. Aliquoting: Prepare single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles. Clearly label each aliquot with date, concentration, and lot number.
4. Storage Conditions: Store working aliquots at -80 °C. Short-term storage at -20 °C may be acceptable for up to the period specified on the datasheet; verify with product documentation.
5. Freeze-Thaw Cycles: Limit freeze-thaw cycles to the minimum necessary. Each additional cycle risks denaturation and loss of motor activity. Addition of carrier protein (e.g., BSA) may be considered if recommended on the datasheet.
6. Contamination Prevention: Use sterile, low-protein-binding tubes and pipette tips. Work in a clean environment to prevent protease contamination.

Research Applications

Recombinant Human CENPE Protein is a versatile research tool applicable across multiple experimental contexts in cell biology, biochemistry, and drug discovery:

• In vitro Motility Assays: CENPE motor domain fragments can be used to study plus-end-directed microtubule motility and to characterize kinesin-7 mechanochemistry.
• Microtubule-Binding Studies: Co-sedimentation assays allow quantification of CENPE–microtubule binding affinity and the effects of regulatory factors or small molecules.
• ATPase Activity Measurement: Colorimetric or fluorometric phosphate-release assays enable characterization of CENPE ATPase kinetics.
• Spindle Assembly Checkpoint Research: CENPE is used to reconstitute SAC signaling cascades in cell-free systems, facilitating mechanistic studies of BubR1 activation and MCC assembly.
• Drug Target Validation: As a validated anticancer target, Recombinant Human CENPE Protein supports high-throughput screening (HTS) and structure-activity relationship (SAR) studies for inhibitor development.
• Protein–Protein Interaction Studies: Pull-down, co-immunoprecipitation, and surface plasmon resonance (SPR) assays can be used to map CENPE interaction networks with kinetochore and checkpoint proteins.

Quality Control & Troubleshooting

Ensuring data quality when working with Recombinant Human CENPE Protein requires attention to both product quality and experimental design:

Quality Control Checkpoints

• Verify protein integrity by running an SDS-PAGE gel on each new lot before use in critical experiments.
• Confirm biological activity using a functional assay (e.g., microtubule co-sedimentation or ATPase assay) if motor activity is required for your application.
• Check the Certificate of Analysis for endotoxin levels if the protein will be used in cell-based assays, as endotoxin contamination can confound results.

Common Issues & Solutions

• Reduced Activity After Storage: May indicate protein degradation due to freeze-thaw cycling. Use freshly thawed single-use aliquots.
• Aggregation or Precipitation: Ensure the protein is reconstituted in the recommended buffer at the correct concentration. Centrifuge briefly at low speed before use to remove any aggregates.
• Non-Specific Bands on SDS-PAGE: May reflect partial degradation or post-translational modification variants; consult the CoA and contact Aladdin Scientific technical support for guidance.
• Low Signal in Binding Assays: Optimize assay buffer conditions including salt concentration, pH, and the presence of nucleotide cofactors (e.g., AMP-PNP or ADP for different microtubule-binding states).

Frequently asked questions

What is the primary function of CENPE in human cells?

CENPE (Centromere Protein E) is a kinesin-7 family motor protein that localizes to the outer kinetochore during mitosis. Its primary functions include driving chromosome congression to the metaphase plate and sustaining spindle assembly checkpoint (SAC) signaling by interacting with checkpoint proteins such as BubR1. Loss of CENPE function leads to chromosome misalignment and aneuploidy.

How should Recombinant Human CENPE Protein be stored to maintain activity?

Recombinant Human CENPE Protein should be stored at -80 °C for long-term preservation of biological activity. Upon receipt, prepare single-use aliquots to avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of motor activity. Always consult the product-specific datasheet and Certificate of Analysis for the recommended storage buffer and any additional stabilization requirements.

What assays are suitable for validating the biological activity of CENPE protein?

Suitable functional assays for Recombinant Human CENPE Protein include microtubule co-sedimentation assays to assess microtubule-binding activity, steady-state ATPase assays (e.g., colorimetric malachite green or NADH-coupled assays) to measure motor ATPase kinetics, and in vitro motility assays using TIRF microscopy to directly observe microtubule gliding. The appropriate assay depends on the specific domain or function under investigation.

Is Recombinant Human CENPE Protein suitable for use as a drug discovery target?

Yes. CENPE has been validated as an anticancer drug target because its inhibition disrupts mitotic chromosome congression and activates the spindle assembly checkpoint, ultimately leading to mitotic arrest and cell death in proliferating cancer cells. Recombinant Human CENPE Protein is therefore suitable for biochemical high-throughput screening (HTS) campaigns and structure-activity relationship (SAR) studies aimed at developing selective kinesin-7 inhibitors.

Can CENPE protein be used in protein–protein interaction studies?

Yes. Recombinant Human CENPE Protein is suitable for protein–protein interaction studies including pull-down assays, co-immunoprecipitation, and biophysical methods such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). These approaches can be used to characterize CENPE interactions with kinetochore components (e.g., CENP-F, Mis12 complex) and checkpoint regulators (e.g., BubR1, BUBR1 kinase domain).

References

1. Yen TJ, et al. CENPE, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1992.
2. Kapoor TM, et al. Chromosomes can congress to the metaphase plate before biorientation. Science. 2006.
3. Mao Y, et al. Cenp-E, a novel centromere-associated protein essential for chromosome alignment. J Cell Biol. 2003.
4. Aladdin Scientific. Recombinant Human CENPE Protein, SKU rp371535. Product Datasheet.

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