Recombinant Danio rerio Protein kinase C beta type (prkcbb), partial

What is the molecular structure and function of Danio rerio prkcbb?

Danio rerio protein kinase C beta b (prkcbb) is a 670 amino acid protein that functions as a calcium-activated and phospholipid-dependent serine/threonine protein kinase . It belongs to the protein kinase C family, which are enzymes that phosphorylate a wide variety of protein targets and participate in diverse cellular signaling pathways. The protein contains characteristic domains including regulatory domains that interact with calcium and diacylglycerol, and a catalytic domain responsible for its kinase activity . In zebrafish, prkcbb plays crucial roles in B-cell receptor signalosome regulation, apoptotic processes, and transcription regulation, paralleling many functions of its mammalian orthologs while exhibiting fish-specific activities in development and physiology.

How does zebrafish prkcbb differ from human PRKCB?

While zebrafish prkcbb and human PRKCB share considerable sequence homology and functional conservation, several notable differences exist in their regulation and tissue-specific functions. Human PRKCB has been extensively characterized in B cell activation, apoptosis induction, endothelial cell proliferation, and intestinal sugar absorption . The zebrafish ortholog maintains many of these functions but displays unique developmental roles specific to teleost biology. Structurally, both proteins function as calcium-activated and phospholipid-dependent kinases, but subtle differences in regulatory domains may confer species-specific activation parameters . When designing cross-species experimental comparisons, researchers should account for these differences, particularly in phosphorylation targets and tissue expression patterns.

What expression systems are optimal for producing recombinant Danio rerio prkcbb?

For recombinant expression of zebrafish prkcbb, several systems have demonstrated success with varying yields and post-translational modifications. Bacterial systems (E. coli) offer cost-effective production but may lack proper folding and post-translational modifications essential for kinase activity. Insect cell systems (Sf9, High Five) provide superior eukaryotic processing capabilities while maintaining reasonable yields. Mammalian expression systems (HEK293, CHO cells) offer the most authentic post-translational modifications but at higher cost and potentially lower yields . When selecting an expression system, researchers should consider the experimental application: structural studies may prioritize quantity and purity, while functional assays require properly folded, active protein with appropriate modifications.

How can I design experiments to investigate prkcbb's role in zebrafish B-cell development?

To investigate prkcbb's role in zebrafish B-cell development, a multi-faceted experimental approach is recommended. Begin with temporal expression analysis using qRT-PCR and in situ hybridization to establish prkcbb expression patterns throughout lymphoid development. CRISPR/Cas9-mediated gene editing can generate prkcbb knockouts or specific domain mutations to assess functional requirements. For cell-specific studies, isolate B-cell populations from transgenic zebrafish lines (e.g., cd79a:GFP) and analyze prkcbb-dependent phosphorylation events using phospho-specific antibodies against known substrates . Rescue experiments using recombinant prkcbb protein microinjection or mRNA can confirm specificity. Comparative analyses between morphant/mutant and wild-type fish should examine B-cell numbers, activation markers, and antibody production following immune challenges.

What are the key signaling pathways interconnected with prkcbb function in zebrafish?

Prkcbb operates within a complex network of signaling pathways in zebrafish. String database analysis reveals strong functional interactions with phospholipase C gamma (plcg1 and plcg2), which generate diacylglycerol and inositol trisphosphate - crucial for prkcbb activation . The mTOR pathway (through direct interaction with mtor protein) connects prkcbb to cellular growth and metabolism regulation . Additional significant interactions include Raf kinases (raf1a, raf1b) linking prkcbb to MAPK signaling cascades controlling cell proliferation and differentiation . The NF-κB pathway activation occurs through prkcbb-mediated phosphorylation of card11/carma1, particularly important in B-cell function . When designing pathway inhibition studies, researchers should consider that prkcbb may function both upstream and downstream of these interacting partners, necessitating careful experimental design with appropriate controls for pathway crosstalk.

How does phosphorylation status affect prkcbb activity and substrate specificity?

Prkcbb activity is intricately regulated by its phosphorylation status, with multiple conserved phosphorylation sites controlling activation state, substrate affinity, and subcellular localization. Three key phosphorylation events are crucial: (1) activation loop phosphorylation in the catalytic domain, which aligns active site residues for catalysis; (2) turn motif phosphorylation, which stabilizes the catalytic core; and (3) hydrophobic motif phosphorylation, which enhances catalytic efficiency. Mass spectrometry analysis of recombinant prkcbb can identify the phosphorylation status at these critical sites. In vitro kinase assays using differentially phosphorylated prkcbb variants demonstrate that phosphorylation pattern dictates substrate preference, with fully phosphorylated enzyme showing broader substrate recognition compared to partially phosphorylated forms . Researchers should ensure consistent phosphorylation status when producing recombinant prkcbb to maintain experimental reproducibility.

What are the optimal methods for purifying recombinant prkcbb while maintaining activity?

Purification of active recombinant prkcbb requires careful consideration of buffer conditions and purification strategy. A recommended approach begins with affinity chromatography using either His-tag or GST-tag systems, depending on the expression construct . Critical buffer components include: (1) mild detergents (0.01-0.05% Triton X-100) to prevent aggregation; (2) reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to maintain thiol groups; (3) phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to preserve phosphorylation status; and (4) protease inhibitors to prevent degradation. Size exclusion chromatography as a polishing step helps remove aggregates that may interfere with activity assays. Throughout purification, maintain temperature at 4°C and minimize freeze-thaw cycles. Activity testing after each purification step allows optimization of conditions that preserve enzymatic function.

How can I accurately assess the kinase activity of recombinant prkcbb?

Multiple complementary approaches can assess recombinant prkcbb kinase activity. Radiometric assays using [γ-32P]ATP provide the gold standard for quantitative activity measurement, detecting phosphate incorporation into model substrates like myelin basic protein or tailored peptides containing consensus recognition sequences. Non-radiometric alternatives include FRET-based peptide sensors that change conformation upon phosphorylation, offering real-time monitoring capabilities. For cellular context assessment, phospho-specific antibodies against known prkcbb substrates can detect phosphorylation events via western blotting or immunofluorescence. When conducting activity assays, critical controls include: (1) heat-inactivated enzyme; (2) kinase-dead mutant (typically K→A mutation in ATP-binding site); and (3) specific PKC inhibitors like Gö6976. Activity should be normalized to protein quantity using Bradford or BCA assays to allow cross-preparation comparisons.

What antibodies and detection methods are validated for zebrafish prkcbb research?

Several antibodies have been validated for zebrafish prkcbb detection, including the polyclonal antibody CSB-PA759675XA01DIL from Cusabio, which targets Danio rerio prkcbb (UniProt ID: Q7SY24) . When selecting antibodies, researchers should consider epitope location: antibodies targeting conserved catalytic domains may cross-react with other PKC family members, while those targeting variable regions offer greater specificity. Complementary detection methods include RNA-based approaches (qRT-PCR, in situ hybridization) for expression analysis and activity-based protein profiling using covalent ATP analogs. For localization studies, fluorescence microscopy using either immunostaining with validated antibodies or expression of fluorescently-tagged prkcbb provides spatial information, though tag interference with function should be verified. Species-specific validation of commercial antibodies is essential, as antibodies raised against mammalian PKC may not recognize zebrafish orthologs with equivalent sensitivity.

How can recombinant prkcbb be utilized in zebrafish neurodevelopmental studies?

Recombinant prkcbb provides a valuable tool for investigating neurodevelopmental processes in zebrafish. Direct microinjection of purified protein or mRNA encoding prkcbb into developing embryos can rescue phenotypes in morphant or mutant models, confirming specificity of observed defects. For mechanistic studies, recombinant protein with site-specific mutations can determine which domains are necessary for neuronal function . Ex vivo applications include treating primary neuronal cultures from zebrafish with recombinant protein to assess effects on neurite outgrowth, axon guidance, and synaptogenesis. Protein-protein interaction studies using pull-down assays with recombinant prkcbb can identify neuronal binding partners specific to zebrafish, potentially revealing teleost-specific signaling mechanisms. When utilizing recombinant protein in developmental studies, researchers should carefully titrate concentrations, as excessive kinase activity may yield non-physiological effects.

What is the significance of prkcbb in modeling human PKC-related pathologies?

Zebrafish prkcbb serves as a valuable model for studying human PKC-related pathologies due to significant functional conservation. Dysregulation of PKC signaling contributes to numerous human diseases including cancer, diabetes, immunological disorders, and neurological conditions . Recombinant prkcbb can be used in pharmaceutical screens to identify compounds that modulate its activity with potential therapeutic applications. Structure-function studies comparing zebrafish prkcbb with human PRKCB can reveal conserved regulatory mechanisms and substrate recognition patterns. When using zebrafish as disease models, researchers should consider both the similarities and differences between fish and human systems. The streamlined genetic background and optical transparency of zebrafish embryos provide advantages for visualizing disease processes in vivo, while the relatively simpler immune system may limit modeling of complex immunological disorders.

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

Several challenges commonly arise when working with recombinant prkcbb. Protein aggregation during expression and purification can be mitigated by optimizing buffer conditions (including mild detergents and stabilizing agents like glycerol) and employing fusion partners that enhance solubility (SUMO, MBP) . Loss of activity during purification often results from dephosphorylation or oxidation; incorporate phosphatase inhibitors and reducing agents throughout all procedures. Inconsistent activity between preparations typically stems from variable phosphorylation states; consider using lambda phosphatase treatment followed by in vitro rephosphorylation with PDK1 to standardize activation status. For long-term storage, avoid repeated freeze-thaw cycles by preparing small aliquots, and include 10-20% glycerol as a cryoprotectant. When troubleshooting expression problems, test multiple tags and positions (N-terminal vs. C-terminal), as tag placement can significantly impact folding and activity.

How should comparative studies between zebrafish prkcbb and mammalian PRKCB be designed and interpreted?

When designing comparative studies between zebrafish prkcbb and mammalian PRKCB, several methodological considerations ensure valid interpretation. Sequence alignment analysis should first identify conserved and divergent regions to guide functional predictions . Expression constructs should maintain equivalent domain structures and regulatory elements. For activity comparisons, standardize assay conditions including temperature (consider that zebrafish proteins may have different temperature optima than mammalian counterparts), substrate concentrations, and cofactor availability. When interpreting functional differences, distinguish between intrinsic protein properties and species-specific cellular contexts by performing cross-species complementation studies. Particularly important is the analysis of activation parameters, as lipid and calcium sensitivities may differ between fish and mammalian orthologs. Publication data should include raw kinetic parameters rather than normalized values to allow direct cross-species comparison by subsequent researchers.

What quality control metrics should be applied to recombinant prkcbb preparations?

Rigorous quality control ensures experimental reproducibility when working with recombinant prkcbb. Purity assessment via SDS-PAGE should demonstrate >90% homogeneity, with verification by mass spectrometry to confirm primary sequence and identify any modifications or truncations. Phosphorylation status analysis using phospho-specific antibodies or mass spectrometry is crucial, as it directly impacts activity . Functional validation through in vitro kinase assays should demonstrate both autophos­phorylation capacity and substrate phosphorylation with expected kinetic parameters. Thermal stability assessment using differential scanning fluorimetry provides information about proper folding and buffer optimization. Before use in complex experiments, verify that the recombinant protein responds appropriately to known activators (phorbol esters, diacylglycerol analogs) and inhibitors (Gö6976, staurosporine). Each protein preparation should include a certificate of analysis documenting these quality metrics to ensure experimental consistency across studies.