Recombinant Mouse Tetratricopeptide repeat protein 22 (Ttc22)

What is the structural characterization of mouse TTC22?

Mouse TTC22 belongs to the tetratricopeptide repeat domain-containing family of proteins, characterized by seven tetratricopeptide repeat motifs. These TPR motifs consist of 34-amino acid sequences that form a helix-turn-helix arrangement, typically mediating protein-protein interactions and chaperone activity in various cellular processes . Mouse TTC22 shares approximately 86% sequence identity with human TTC22, particularly in the amino acid region 359-448, which is often used as a control fragment in experimental settings . The protein's functional domains include multiple TPR repeats that fold into a superhelical structure, creating binding pockets for partner proteins. Alternative spliced transcript variants have been documented for this gene, potentially expanding its functional diversity in different cellular contexts .

What are the expression patterns of TTC22 in mouse tissues?

TTC22 exhibits distinctive tissue-specific expression patterns across mouse developmental stages and adult tissues. Based on gene expression profiling data, TTC22 expression can be quantified in various tissues and compared to baseline expression levels . The protein shows varying expression levels across different brain regions according to the Allen Brain Atlas database, suggesting tissue-specific functions . Expression analysis reveals that TTC22 may exhibit differential regulation during development compared to adult tissues. TTC22 expression has been documented in multiple cell types, with varying levels that correlate with specific cellular states or differentiation stages. This expression pattern suggests potential roles in tissue-specific functions that may be developmentally regulated or context-dependent .

How should mouse TTC22 recombinant proteins be stored and handled for optimal stability?

For optimal stability, recombinant mouse TTC22 proteins should be stored at -20°C and repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of activity . When working with recombinant TTC22, it's recommended to prepare small aliquots before freezing to minimize the number of freeze-thaw cycles per sample. The protein is typically supplied in a buffer containing 1M urea and PBS at pH 7.4 without preservatives, which maintains stability while preventing aggregation . When thawing the protein, it should be done gradually on ice rather than at room temperature to prevent denaturation. For experiments requiring longer handling times, maintaining the protein at 4°C during the experimental procedure is advised rather than at room temperature, which can accelerate degradation.

How can you verify the purity and identity of recombinant mouse TTC22?

Verification of recombinant mouse TTC22 purity and identity involves multiple complementary analytical techniques. SDS-PAGE analysis followed by Coomassie blue staining should show a single band at the expected molecular weight, with purity typically exceeding 80% for research-grade preparations . Western blotting using specific anti-TTC22 antibodies provides confirmation of protein identity, while mass spectrometry analysis can verify the exact molecular weight and sequence coverage. For His-tagged recombinant TTC22, immuno-detection using anti-His antibodies provides additional confirmation of the recombinant protein's identity . Functional assays measuring protein-protein interaction capabilities characteristic of TPR-containing proteins can further validate proper folding and biological activity of the preparation.

What roles does TTC22 play in disease models, particularly cancer?

TTC22 has emerged as a potential prognostic marker and therapeutic target in cancer research, particularly in pancreatic adenocarcinoma models. Studies reveal that TTC22 expression negatively correlates with survival prognosis in pancreatic cancer patients, suggesting its involvement in disease progression . Functional enrichment analysis has demonstrated a significant correlation between TTC22 expression and immune infiltration in the tumor microenvironment, with TTC22 appearing to inhibit tumor immunity and showing negative correlation with plasmacytoid dendritic cells . Molecular mechanism investigations suggest TTC22 may influence oncogenic signaling pathways that regulate tumor cell proliferation and metastasis. Knockdown experiments using siRNAs targeting TTC22 in pancreatic cancer cell lines (PANC-1 and PaTu8988) have been employed to elucidate its functional role in cancer cell behavior, providing insights into potential therapeutic approaches .

How can researchers effectively design knockdown experiments for mouse TTC22?

Designing effective knockdown experiments for mouse TTC22 requires careful consideration of several methodological aspects. Start by designing multiple siRNA constructs targeting different regions of the TTC22 transcript, as exemplified in pancreatic cancer studies where researchers utilized three distinct siRNA sequences to ensure robust knockdown . Transfection optimization is crucial; for adherent cell lines, lipid-based transfection reagents like Lipofectamine have proven effective, with serum-free DMEM recommended during the transfection process . Knockdown efficiency should be quantified at both mRNA level using qRT-PCR and protein level using Western blotting with specific anti-TTC22 antibodies, with 70-90% reduction considered sufficient for functional studies. To control for off-target effects, include both a negative control siRNA (scrambled sequence) and validate phenotypes with multiple independent siRNA constructs targeting different regions of TTC22. Post-knockdown functional assays should be carefully selected based on the hypothesized role of TTC22, potentially including proliferation, migration, invasion, and protein-protein interaction studies.

What is the evolutionary significance of TPR motifs in TTC22 compared to other TPR-containing proteins?

The evolutionary history of TTC22's TPR motifs reflects a fascinating case of protein evolution through repeat amplification. TPR domains, including those in TTC22, likely originated from the amplification of subdomain-sized peptides that initially required RNA scaffolding to assume their active conformation . Research suggests that ancestral helical hairpins, similar to those found in ribosomal protein S20 (RPS20), could form TPR-like structures when amplified with just 2-5 point mutations per repeat . These mutations appear neutral in the parent organism, suggesting they could have been sampled during evolution without adverse effects. The structural transition from non-folding peptides to folded repeat proteins demonstrates how repetition enables peptides incapable of independent folding to yield structured proteins, a principle applicable to TTC22's evolutionary development . Comparative analysis between TTC22 and other TPR-containing proteins reveals conservation patterns of key structural residues at specific positions that maintain the characteristic helix-turn-helix arrangement while allowing functional diversification.

How can mouse TTC22 be used in protein-protein interaction studies?

Mouse TTC22, with its seven tetratricopeptide repeats, serves as an excellent subject for protein-protein interaction studies due to TPR domains' established role in mediating molecular interactions. For in vitro binding assays, researchers can employ purified recombinant TTC22 (preferably with fusion tags like His or GST for purification) immobilized on appropriate matrices to identify binding partners from cell lysates, followed by mass spectrometry identification . Yeast two-hybrid screening represents another powerful approach, using TTC22 as bait to screen against mouse cDNA libraries to identify novel interacting proteins. Co-immunoprecipitation experiments in relevant mouse cell lines can validate interactions in a more physiological context, using either antibodies against endogenous TTC22 or epitope-tagged overexpressed constructs . Advanced methods like bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) allow real-time monitoring of TTC22 interactions in living cells, providing insights into the dynamics and subcellular localization of these interactions.

What are the best practices for using recombinant TTC22 in blocking experiments?

When using recombinant mouse TTC22 in blocking experiments, several methodological considerations ensure optimal results. The blocking protocol should employ a 100-fold molar excess of the protein fragment control relative to the corresponding antibody, calculated based on concentration and molecular weight . Pre-incubation of the antibody-protein control fragment mixture should be conducted for approximately 30 minutes at room temperature to allow sufficient binding . For Western blotting applications, the blocked antibody solution should be used in place of the primary antibody solution, maintaining the same incubation time and temperature as would be used for the unblocked antibody. In immunohistochemistry and immunocytochemistry blocking experiments, researchers should include additional controls such as unblocked antibody and isotype controls to distinguish specific signal reduction from background. Optimization may be required for each specific application, with titration of the blocking protein concentration potentially improving specificity while preserving necessary sensitivity.

How can researchers analyze TTC22 expression in relation to immune cell infiltration?

Analysis of TTC22 expression in relation to immune cell infiltration requires integrated bioinformatic and experimental approaches. The ESTIMATE algorithm can be employed to calculate stromal and immune scores from gene expression data, providing a quantitative measure of immune cell presence in relation to TTC22 expression levels . Single sample Gene Set Enrichment Analysis (ssGSEA) using the R package GSVA allows researchers to annotate 24 immune-cell markers to determine specific immune infiltrates, providing granular information about which immune cell types correlate with TTC22 expression . Statistical analysis using Spearman's correlation can quantify the relationship between TTC22 expression and various immune cell populations, while Wilcoxon's rank-sum test can assess differences in immune cell infiltration between high and low TTC22 expression groups . Flow cytometry validation in mouse tumor models provides experimental confirmation of bioinformatic predictions, analyzing the prevalence of specific immune cell populations in tumors with varying TTC22 expression levels.

What experimental approaches can determine TTC22's impact on cellular signaling pathways?

Multiple experimental approaches can elucidate TTC22's impact on cellular signaling pathways in mouse models. RNA sequencing following TTC22 knockdown or overexpression in relevant cell lines can identify differentially expressed genes, providing insights into affected pathways through subsequent Gene Ontology and KEGG pathway analysis . Phosphoproteomics analysis comparing control and TTC22-modulated cells reveals changes in protein phosphorylation status, highlighting directly affected signaling nodes. Western blotting for key phosphorylated signaling molecules (e.g., MAPKs, AKT, STATs) following TTC22 modulation provides targeted validation of hypothesized pathway connections. Reporter assays using luciferase constructs controlled by response elements for specific transcription factors can measure pathway activation levels in response to TTC22 manipulation. Co-immunoprecipitation followed by mass spectrometry identifies TTC22 binding partners within signaling complexes, elucidating its direct role in signaling networks.

How does mouse TTC22 compare structurally and functionally to human TTC22?

Mouse and human TTC22 share significant structural and functional similarities with some notable differences. Sequence alignment reveals approximately 86% identity between mouse and human TTC22, particularly in the 359-448 amino acid region, indicating high evolutionary conservation of functional domains . The seven tetratricopeptide repeat motifs characteristic of both proteins maintain similar spacing and organization, suggesting conservation of protein-protein interaction capabilities between species. Expression pattern comparison shows similar tissue distribution with some species-specific differences in expression levels, potentially reflecting subtle functional adaptations. While both proteins share core functions in mediating protein-protein interactions, species-specific binding partners may exist due to sequence variations outside the highly conserved TPR domains. These molecular differences should be considered when translating findings between mouse models and human clinical applications, particularly when developing therapeutic strategies targeting TTC22.

How can researchers design effective TTC22 overexpression systems for functional studies?

Designing effective TTC22 overexpression systems requires careful consideration of expression vectors, cell types, and experimental controls. Select appropriate expression vectors based on research goals—mammalian expression vectors with strong promoters (CMV, EF1α) for high expression, or inducible systems (Tet-On/Off) for controlled expression timing . Include epitope tags (FLAG, HA, His) or fluorescent protein fusions (GFP, mCherry) for detection and purification, positioned to minimize interference with TTC22 function, ideally at the C-terminus to avoid disrupting the N-terminal protein-protein interaction domains. When selecting cell lines, prioritize those with low endogenous TTC22 expression to maximize the signal-to-background ratio, and consider species compatibility (mouse cells for mouse TTC22) to ensure proper post-translational modifications and protein-protein interactions. Essential controls should include empty vector transfections, inactive TTC22 mutants (particularly mutations in key TPR residues), and monitoring of expression levels across experiments to account for expression-dependent effects.

How should researchers interpret contradictory findings about TTC22 function?

When encountering contradictory findings regarding TTC22 function, researchers should employ a systematic approach to resolution and interpretation. Context dependency analysis is crucial—TTC22 may exhibit different functions in different tissue types, developmental stages, or disease states, as evidenced by its variable expression patterns across tissues documented in the Allen Brain Atlas and other databases . Technical considerations must be evaluated, including differences in experimental models (in vitro vs. in vivo), knockdown/overexpression methods, and assay conditions that might explain apparently contradictory results. Post-translational modifications and splice variants can dramatically alter protein function, so researchers should determine which TTC22 isoforms are being studied in conflicting reports . Protein interactome differences may explain functional variation, as TTC22's activity is likely mediated through protein-protein interactions that differ across cellular contexts. Resolution strategies should include independent validation using multiple approaches (genetic knockdown, overexpression, domain mutations) and reconciliation attempts through unified models that accommodate context-dependent functions.

What computational methods best predict TTC22 structure-function relationships?

Predicting TTC22 structure-function relationships requires specialized computational methods tailored to repeat-containing proteins. Homology modeling using solved structures of TPR-containing proteins as templates provides insights into the three-dimensional arrangement of TTC22's TPR repeats, with particular attention to the superhelical structure formed by the repeating helix-turn-helix motifs . Molecular dynamics simulations can reveal the flexibility of TPR motifs and identify potential binding pockets, with extended simulations (>100ns) necessary to capture the dynamic behavior of repeat proteins. Machine learning approaches trained on known TPR-protein interactions can predict potential binding partners based on sequence features and surface properties. Evolutionary coupling analysis identifies co-evolving residues within TPR repeats that maintain structural integrity or participate in protein-protein interactions. Integrative modeling combining experimental data (limited proteolysis, crosslinking, SAXS) with computational predictions provides the most comprehensive structural models, capturing both the repeat architecture and functional interactions of TTC22.

Research Applications Table