CKB Human, Pichia

What is the molecular structure of CKB Human produced in Pichia pastoris?

CKB Human Recombinant produced in Pichia pastoris is a dimeric glycosylated full-length polypeptide chain comprised of two identical B subunits. The protein has a molecular weight of approximately 47 kDa and maintains an amino acid sequence identical to the native enzyme. The protein is typically purified under non-denaturing conditions to preserve its native conformation and biological activity .

What are the optimal storage conditions for CKB Human recombinant protein?

For maximum stability and retention of enzymatic activity, CKB Human recombinant protein should be stored below -18°C. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of activity. The typical formulation includes 10mM Bis-Tris-HCl pH-6.0, 50% glycerol, 0.5mM EDTA, and 0.5mM DTT, which helps maintain protein stability during storage . For researchers planning long-term experiments, it is advisable to prepare single-use aliquots upon receipt to minimize freeze-thaw damage.

How can CKB Human be used in asthma research models?

Recent research has identified an inverse association between circulating creatine kinase levels and childhood asthma. Studies across multiple birth cohorts demonstrated lower concentrations of circulating CK and reduced whole blood CKM and CKB expression in asthma patients compared to control subjects . For experimental design, researchers could:

• Use purified CKB to examine enzyme activity in normal versus asthmatic clinical samples

• Compare enzymatic kinetics of CKB in respiratory tissue homogenates

• Develop in vitro models using airway epithelial cells with varying CKB expression levels

• Investigate the effects of CKB inhibitors on airway hyperresponsiveness

Mouse models have shown that CKB inhibition blocks the resolution of airway hyperresponsiveness and reduction of airway mucin following allergen challenge, suggesting a potential mechanistic role of CKB in asthma pathophysiology .

What methodological approaches can be used to analyze CKB function in energy metabolism studies?

To investigate CKB's role in cellular energy metabolism, researchers can employ several methodological approaches:

• Enzymatic Activity Assays: Measure CKB activity using spectrophotometric methods that track the formation of creatine from phosphocreatine

• Cellular Bioenergetics: Use Seahorse XF analyzers to measure oxygen consumption rates and extracellular acidification rates in cells with varying CKB expression

• Phosphocreatine Shuttle Analysis: Employ 31P-NMR spectroscopy to track real-time phosphocreatine/ATP ratios

• Subcellular Localization Studies: Perform immunofluorescence microscopy using anti-CKB antibodies to determine the intracellular distribution of CKB

For experiments requiring detection of CKB, validated antibodies like Picoband anti-CKB (A01695-1) have demonstrated efficacy in ELISA, Western blot, and immunohistochemistry applications across human, mouse, and rat samples .

How does CKB function differently from other creatine kinase isoforms in cellular energy dynamics?

CKB functions as a brain-type creatine kinase that forms homodimers (BB) in neural tissues and heterodimers (MB) with muscle-type CK in cardiac tissue. The functional differences include:

CKB plays a crucial role in tissues with fluctuating energy demands, particularly in neural tissues where it helps maintain ATP levels during periods of high metabolic activity. Unlike muscle-specific isoforms, CKB demonstrates unique regulatory properties and substrate affinities optimized for neural tissue energy homeostasis .

What factors influence the expression and activity of recombinant CKB in Pichia pastoris systems?

Several factors can significantly impact the expression and activity of recombinant CKB in Pichia pastoris:

• Promoter selection: The AOX1 promoter is commonly used for methanol-induced expression, while constitutive promoters like GAP may provide continuous expression

• Codon optimization: Adapting the human CKB sequence to Pichia codon bias can enhance expression levels

• Signal sequence: Selection of appropriate secretion signals affects protein processing and yield

• Cultivation conditions:

  • Temperature (typically 28-30°C is optimal)

  • pH (maintaining pH 5-6 during growth phase)

  • Dissolved oxygen (keeping above 20% saturation)

  • Carbon source concentration (methanol feeding strategy for AOX1 promoter)

• Post-translational modifications: Hyperglycosylation can occur in Pichia, potentially affecting protein function

Researchers should optimize these parameters through Design of Experiments (DoE) approaches to maximize both yield and activity of the recombinant CKB .

What is the role of CKB in cell-free protein synthesis systems?

• Creatine phosphate as the primary energy source

• Creatine kinase to catalyze the transfer of phosphate from creatine phosphate to ADP, regenerating ATP

• Additional cofactors like DTT, magnesium, and potassium

For researchers developing CFPS systems, it's important to note that the rapid depletion of energy-regeneration components significantly impacts protein synthesis duration and yield. When using HEK293-derived systems, researchers have achieved protein yields up to 300 μg/ml without requiring exogenous CK, suggesting that endogenous CK activity in the cell extract may be sufficient .

What strategies can resolve issues with low activity of Pichia-expressed CKB?

When facing low activity issues with Pichia-expressed CKB, consider these methodological approaches:

• Expression optimization:

  • Verify the integrity of the expression construct

  • Test different Pichia strains (X-33, GS115, KM71H)

  • Optimize induction conditions (methanol concentration, temperature, duration)

• Purification refinement:

  • Ensure purification under non-denaturing conditions

  • Include stabilizing agents (glycerol, reducing agents) in all buffers

  • Consider mild detergents if aggregation is observed

• Activity assessment:

  • Verify proper dimer formation via size-exclusion chromatography

  • Assess protein folding using circular dichroism

  • Optimize assay conditions (pH, temperature, buffer composition)

  • Include known activators in activity assays

• Storage optimization:

  • Maintain protein at -20°C or below

  • Include 50% glycerol and reducing agents in storage buffer

  • Aliquot to avoid freeze-thaw cycles

How can researchers validate CKB antibody specificity across different experimental platforms?

To ensure antibody specificity when studying CKB across different experimental platforms, researchers should implement a multi-faceted validation approach:

• Western blot validation:

  • Confirm single band at expected molecular weight (~43 kDa for monomer, ~47 kDa for glycosylated form)

  • Include positive controls (brain tissue lysate) and negative controls (CKB knockout samples)

  • Perform peptide competition assays to confirm specificity

• Immunohistochemistry controls:

  • Use known CKB-positive tissues (brain, kidney) as positive controls

  • Include isotype controls to assess non-specific binding

  • Test antibody in CKB knockout tissues if available

  • Validate tissue-specific staining patterns against literature descriptions

• Cross-reactivity assessment:

  • Test against recombinant CKM to ensure isoform specificity

  • If using across species, verify sequence homology in the epitope region

  • For primate studies, note that antibodies like A01695-1 may cross-react with monkey tissues based on sequence similarity

• Preabsorption controls:

  • Preincubate antibody with purified recombinant CKB before application

  • Specific signal should be significantly reduced after preabsorption

How can CKB expression and activity be leveraged in neurological disease research?

CKB's prominent expression in brain tissue makes it particularly relevant for neurological disease research:

• Neurodegenerative disorders: CKB plays a crucial role in maintaining energy homeostasis in neurons. Researchers can investigate:

  • Changes in CKB expression and activity in Alzheimer's, Parkinson's, and Huntington's disease models

  • The role of CKB in protecting neurons from excitotoxicity and oxidative stress

  • Using purified CKB to study potential post-translational modifications in disease states

• Traumatic brain injury (TBI) and stroke:

  • Measure CKB release as a biomarker of neural damage

  • Investigate the neuroprotective potential of enhanced CKB activity

  • Develop therapeutic strategies targeting the creatine/phosphocreatine system

• Methodological approaches:

  • Use Pichia-expressed CKB in enzyme replacement therapy models

  • Develop cell-penetrating CKB variants for experimental delivery

  • Create CKB activity modulators to test in disease models

• Therapeutic potential exploration:

  • Screen for small molecule enhancers of CKB activity

  • Test creatine supplementation combined with CKB modulation

  • Investigate CKB gene therapy approaches for neurological conditions

What insights from CKB research in asthma models could be applied to other inflammatory conditions?

The association between CKB and asthma suggests broader implications for inflammatory conditions:

• Mechanistic parallels:

  • The finding that CKB inhibition blocks resolution of airway hyperresponsiveness suggests CKB may play a role in resolving inflammation

  • Similar mechanisms might operate in other inflammatory conditions like rheumatoid arthritis, inflammatory bowel disease, or psoriasis

• Research approaches:

  • Measure CKB expression in tissue samples from patients with various inflammatory conditions

  • Investigate CKB's role in cellular energy dynamics during inflammation resolution

  • Study the relationship between inflammatory mediators and CKB expression/activity

  • Develop animal models to test whether CKB enhancement accelerates resolution of inflammation

• Experimental design considerations:

  • Use recombinant CKB to study enzyme kinetics in inflammatory versus normal tissue environments

  • Employ CKB inhibitors like BU99006 in models of inflammation resolution

  • Investigate time-course of CKB expression changes during inflammatory progression and resolution