Recombinant Rat T-complex protein 11 homolog (Tcp11)

What is Recombinant Rat T-complex protein 11 homolog (Tcp11)?

Recombinant Rat Tcp11 is a laboratory-produced version of the t-complex protein 11 homolog, a protein naturally encoded by the Tcp11 gene in rats. The native protein is homologous to the mouse t-complex protein 11, with the full-length rat version consisting of 566 amino acids . Recombinant variants are produced in various expression systems including E. coli, mammalian cells (particularly HEK293), and other platforms to facilitate research applications . These recombinant proteins are typically tagged with affinity markers such as His, Avi, or Fc tags to enable purification and detection in experimental systems .

What is the molecular structure and known function of Tcp11?

Tcp11 is characterized as a protein with multiple functional domains, though its complete three-dimensional structure remains to be fully elucidated. In humans, the TCP11 gene encodes a protein that spans 503 amino acids, while the rat homolog is slightly larger at 566 amino acids . The protein appears to play roles in cellular processes including proliferation and migration as evidenced by research in cancer cell lines . Recent studies have indicated that TCP11 overexpression in human cervical cancer cells can inhibit proliferation and promote apoptosis, suggesting a potential tumor suppressor function in certain contexts .

How does Tcp11 expression vary across tissues?

While comprehensive tissue expression profiling data for rat Tcp11 is limited in the provided materials, research indicates that the TCP11 gene shows differential expression patterns in normal versus cancerous tissues. For instance, studies using the GEPIA database have revealed that TCP11 is highly expressed in cervical cancer tissues compared to normal cervical tissues . This differential expression pattern suggests tissue-specific roles for Tcp11 that may be important for understanding its biological function in different physiological and pathological contexts.

What expression systems are optimal for producing functional Recombinant Rat Tcp11?

Multiple expression systems have been successfully employed to produce recombinant Rat Tcp11, each with distinct advantages depending on research requirements:

For applications requiring full-length protein with authentic post-translational modifications, mammalian expression systems such as HEK293 cells are preferable . When larger quantities of protein are needed for structural studies or initial screening, E. coli-based systems may be more appropriate despite potential limitations in post-translational modifications .

How can researchers validate the functionality of recombinant Rat Tcp11?

Validation of recombinant Rat Tcp11 functionality should employ multiple complementary approaches:

• Structural integrity assessment: SDS-PAGE, Western blotting, and mass spectrometry to confirm protein size and purity.

• Functional assays: Based on the emerging understanding of Tcp11's role in cell proliferation and apoptosis, researchers can design cell-based assays to evaluate:

  • Effects on cell proliferation (using methods such as MTT/XTT assays)

  • Impact on cell cycle progression (using flow cytometry)

  • Influence on apoptosis pathways (measuring caspase-3 activation and PARP cleavage)

• Binding partner identification: Co-immunoprecipitation or pull-down assays to verify interactions with known or predicted binding partners.

The choice of validation methods should be guided by the specific hypothesis being tested and the intended application of the recombinant protein.

How should experiments be designed to evaluate Tcp11 function in cellular models?

When designing experiments to evaluate Tcp11 function, researchers should follow systematic experimental design principles:

• Define clear variables: Identify independent variables (e.g., Tcp11 expression levels) and dependent variables (e.g., cell proliferation rate, apoptosis markers) .

• Formulate specific hypotheses: For example, "Overexpression of Tcp11 in rat cell lines will decrease proliferation rates by X% compared to control cells" .

• Include appropriate controls: Empty vector controls, wild-type protein controls, and dose-response relationships should be established .

• Select appropriate cell models: Consider the endogenous expression of Tcp11 in candidate cell lines and choose models relevant to the biological context being studied.

• Measure multiple endpoints: Assess effects on cell cycle (using markers like CDK1 and Cyclin B1), apoptosis (caspase-3, cleaved-PARP), and other relevant cellular processes such as EMT (ZO-1, E-cadherin) based on research showing Tcp11's involvement in these pathways .

What are the optimal approaches for overexpression and knockdown studies of Tcp11?

For manipulation of Tcp11 expression in experimental systems:

Overexpression strategies:

• Lentiviral vectors have been successfully employed to overexpress TCP11 in cell lines, with selection using puromycin to establish stable cell lines .

• When using tagged versions, consider that the addition of tags (such as 3×Flag tags) will increase the molecular weight of the protein (e.g., from 503 to 527 amino acids for human TCP11) .

Knockdown/knockout approaches:

• siRNA approaches have been validated for TCP11 knockdown in human cell lines .

• For rat models, similar RNA interference approaches can be adapted, targeting rat-specific sequences.

• CRISPR-Cas9 genome editing offers another approach for complete knockout studies.

For either approach, expression changes should be quantified at both mRNA level (using qRT-PCR) and protein level (using Western blot) to confirm successful manipulation .

How can researchers investigate the signaling pathways influenced by Tcp11?

To investigate signaling pathways involved with Tcp11:

• Proteomics approaches: Use mass spectrometry-based methods following immunoprecipitation to identify binding partners and post-translational modifications.

• Phosphorylation studies: Examine how Tcp11 phosphorylation status changes under different cellular conditions, and how this affects its function.

• Transcriptomics: RNA-seq analysis comparing wild-type vs. Tcp11 overexpression or knockdown can reveal downstream gene expression changes.

• Pathway analysis: Based on existing research showing Tcp11's effects on cell cycle and apoptosis markers, focused analysis of these pathways using specific inhibitors or activators can help place Tcp11 within known signaling networks .

• Protein localization: Immunofluorescence studies to track Tcp11 subcellular localization in response to different stimuli.

What are the current contradictions in the research literature regarding Tcp11 function?

A notable contradiction in the current literature involves TCP11's role in cancer biology. While the research presented in search result indicates that TCP11 overexpression inhibits proliferation and migration of cervical cancer cells (suggesting a tumor suppressor role), the same study notes that TCP11 is actually highly expressed in cervical cancer tissues compared to normal tissues . This paradoxical finding suggests context-dependent functions that require further investigation.

Another area of controversy may involve species-specific differences in Tcp11 function, as the protein sequence and possibly function vary between human (503 amino acids) and rat (566 amino acids) versions .

What are common challenges in working with recombinant Rat Tcp11 and how can they be addressed?

Common challenges include:

• Protein solubility issues: If insolubility occurs with E. coli-expressed protein, consider:

  • Optimizing induction conditions (temperature, IPTG concentration)

  • Using solubility-enhancing fusion tags

  • Switching to mammalian expression systems

• Low expression yields: May be addressed by:

  • Codon optimization for the expression host

  • Using stronger promoters

  • Optimizing culture conditions

• Functional inactivity: If the recombinant protein lacks expected activity:

  • Verify proper folding using circular dichroism

  • Ensure critical post-translational modifications are present

  • Consider the impact of purification methods on protein structure

What are the recommended storage conditions for maintaining recombinant Rat Tcp11 stability?

While specific storage conditions for Rat Tcp11 are not detailed in the provided materials, general best practices for recombinant proteins include:

• Short-term storage (1-2 weeks): 4°C in appropriate buffer with protease inhibitors

• Medium-term storage (1-6 months): -20°C in single-use aliquots

• Long-term storage (>6 months): -80°C with cryoprotectants such as glycerol (10-50%)

Buffer composition should be optimized based on the specific properties of the recombinant Rat Tcp11 preparation, potentially including stabilizing agents and avoiding repeated freeze-thaw cycles.

How is Tcp11 being investigated in disease models beyond cancer?

While the search results primarily highlight TCP11's role in cervical cancer , the protein's expression across diverse tissues suggests potential roles in other physiological and pathological processes. Future research may explore:

• Reproductive biology: Given the testis-specific designation in some annotations , investigation of Tcp11's role in male fertility and reproductive disorders represents a logical research direction.

• Developmental biology: Examining the temporal expression patterns of Tcp11 during embryonic and postnatal development could reveal roles in specific developmental processes.

• Neurological disorders: The t-complex, originally identified in mice, has diverse neurological implications that might be relevant to Tcp11 function in the nervous system.

What novel technologies are advancing our understanding of Tcp11 function?

Emerging technologies that could enhance Tcp11 research include:

• Cryo-EM and advanced structural biology: Determining the three-dimensional structure of Tcp11 and its complexes.

• Single-cell analysis: Examining Tcp11 expression and function at single-cell resolution to uncover cell type-specific roles.

• Organoid models: Using three-dimensional tissue culture systems to study Tcp11 in more physiologically relevant contexts.

• In vivo imaging: Developing tools to visualize Tcp11 localization and dynamics in living cells and organisms.