Recombinant Clostridium perfringens UPF0397 protein CPF_1836 (CPF_1836)

How is recombinant CPF_1836 typically produced for research applications?

Recombinant CPF_1836 is typically produced using in vitro E. coli expression systems, with an N-terminal 10xHis-tag to facilitate purification. The expression region spans positions 1-185 of the native protein . The standard production method involves:

• Cloning the CPF_1836 gene into an appropriate expression vector

• Transformation into competent E. coli cells

• Induction of protein expression

• Cell lysis and protein extraction

• Purification via nickel affinity chromatography utilizing the His-tag

• Formulation in appropriate buffer systems

The final product is typically supplied either as a liquid formulation or as lyophilized powder in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . This preparation provides stability while maintaining structural integrity for experimental applications.

What are the optimal storage conditions for maintaining CPF_1836 stability?

For optimal stability, recombinant CPF_1836 should be stored at -20°C/-80°C upon receipt, with aliquoting recommended to avoid repeated freeze-thaw cycles, which can compromise protein integrity. The shelf life varies depending on storage conditions:

Working aliquots may be stored at 4°C for up to one week . The stability is influenced by multiple factors including buffer composition, storage temperature, and the intrinsic properties of the protein itself. For proteins with transmembrane domains like CPF_1836, addition of stabilizing agents such as glycerol or trehalose can significantly extend functional lifetime.

What experimental controls are essential when working with recombinant CPF_1836 in functional assays?

When designing functional assays with recombinant CPF_1836, the following controls are essential:

Additionally, when incorporating CPF_1836 into cellular assays, researchers should include appropriate cellular controls such as untransfected cells and cells expressing irrelevant proteins of similar size and localization. In animal models, control groups receiving sterile, non-toxic culture medium (as used in the enterotoxemia model) are essential for result interpretation .

How can researchers investigate potential interactions between CPF_1836 and other C. perfringens virulence factors?

To investigate potential interactions between CPF_1836 and other virulence factors, researchers can employ several complementary approaches:

• Co-expression Analysis: Analyze genomic data to identify genes co-expressed with CPF_1836 across different conditions, potentially indicating functional relationships. Whole genome sequence (WGS) phylogenomic reconstruction approaches similar to those used in C. perfringens foodborne outbreak studies could reveal evolutionary relationships between CPF_1836 and characterized virulence genes .

• Protein-Protein Interaction Assays:

  • Pull-down assays using His-tagged CPF_1836 as bait

  • Bacterial two-hybrid systems

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Cross-linking mass spectrometry to identify interacting partners

• Functional Studies:

  • Co-introduction of CPF_1836 with known toxins in cellular models

  • Evaluation of synergistic effects through quantitative virulence assays

Historical precedent from C. perfringens research suggests that virulence often involves complex interactions between multiple factors. For example, studies have shown heterogeneity in virulence gene profiles even within outbreaks, with two or three different profiles detected in some foodborne outbreak investigations . This highlights the importance of considering potential cooperation between CPF_1836 and established virulence factors.

What are the recommended protocols for structural characterization of CPF_1836's transmembrane domains?

For comprehensive structural characterization of CPF_1836's transmembrane domains, researchers should employ multiple complementary techniques:

• Computational Analysis:

  • TMHMM, HMMTOP, or Phobius for transmembrane prediction

  • Molecular dynamics simulations in lipid bilayers

  • 3D structure prediction using AlphaFold or similar platforms

• Experimental Approaches:

  • Circular Dichroism (CD) spectroscopy to determine secondary structure content

  • NMR spectroscopy with detergent-solubilized protein

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to identify exposed regions

• Functional Mapping:

  • Alanine scanning mutagenesis of predicted transmembrane residues

  • Accessibility studies using cysteine labeling

  • Fluorescence spectroscopy with environment-sensitive probes

The complete amino acid sequence reveals multiple hydrophobic regions consistent with transmembrane segments (e.g., "VAIGIGSAVFMILGRFGSLPTGIPNT" and other similar stretches) . These regions likely adopt alpha-helical conformations that span the bacterial membrane, while intervening hydrophilic segments form loops on either side of the membrane.

How can researchers differentiate between specific and non-specific effects when evaluating CPF_1836 in host-pathogen interaction models?

Differentiating specific from non-specific effects in host-pathogen interaction studies with CPF_1836 requires rigorous experimental design:

• Dose-Response Relationships:

  • Establish clear dose-response curves

  • Determine EC50/IC50 values for specific effects

  • Compare concentration thresholds between specific and non-specific responses

• Molecular Specificity Controls:

  • Structure-function analysis through targeted mutations

  • Competition assays with excess unlabeled protein

  • Blocking experiments with antibodies against specific domains

• Temporal Resolution:

  • Time-course experiments to distinguish immediate from delayed effects

  • Pulse-chase approaches to track protein internalization and processing

• Cross-Validation Approaches:

  • Parallel testing in multiple cell/tissue types

  • Comparison with related proteins from non-pathogenic species

  • Correlation of in vitro findings with in vivo observations

When interpreting results, researchers should consider the heterogeneity observed in C. perfringens strains. Studies have shown that multiple different virulence gene profiles can be associated with a single outbreak , suggesting complex pathogenicity mechanisms that may include both specific and non-specific effects.

What advanced genomic approaches can elucidate the evolutionary context of CPF_1836 within Clostridium species?

To investigate the evolutionary context of CPF_1836, researchers can apply several advanced genomic approaches:

• Comparative Genomics:

  • Pan-genome analysis across Clostridium species

  • Identification of CPF_1836 homologs in related bacteria

  • Synteny analysis to examine conservation of genomic context

• Phylogenomic Reconstruction:

  • Core-genome single nucleotide polymorphism (SNP) analysis

  • Maximum likelihood phylogenetic tree construction

  • Ancestral sequence reconstruction

• Selection Pressure Analysis:

  • Calculation of dN/dS ratios to identify selection signatures

  • Identification of conserved vs. variable regions

  • Coevolution analysis with interacting partners

• Horizontal Gene Transfer Assessment:

  • Anomalous GC content or codon usage analysis

  • Identification of mobile genetic elements in proximity

  • Comparison with phylogenetic patterns of housekeeping genes

These approaches can be modeled after those used in large-scale genomic analyses of C. perfringens strains, where whole genome sequence phylogenomic reconstruction based on core-genome SNPs has successfully identified distinct clades that correlate with virulence characteristics . For example, such analyses have distinguished chromosomal cpe-positive strains from cpe-negative and plasmid-borne cpe strains, revealing unexpected evolutionary relationships.

How might CPF_1836 contribute to Clostridium perfringens pathogenesis based on its predicted structure and location?

Based on structural predictions and transmembrane localization, CPF_1836 may contribute to C. perfringens pathogenesis through several potential mechanisms:

• Membrane Integrity and Stress Response:
  As a transmembrane protein, CPF_1836 likely contributes to bacterial membrane structure and function. Its hydrophobic regions (e.g., "IGFIGHALKDIVFFGSPWISW" and similar sequences) suggest potential roles in maintaining membrane integrity during environmental stress conditions encountered during host infection.

• Transport Functions:
  The predicted transmembrane topology suggests possible involvement in:

  • Ion transport or homeostasis

  • Nutrient acquisition during infection

  • Export of virulence factors or toxins

• Signaling and Environmental Sensing:
  Transmembrane proteins often function in signal transduction pathways that:

  • Detect environmental cues in the host

  • Trigger expression of virulence factors

  • Coordinate bacterial responses to host defenses

• Adhesion and Host Interaction:
  Surface-exposed regions may participate in:

  • Direct interactions with host cell receptors

  • Biofilm formation during infection

  • Evasion of host immune responses

Studies of C. perfringens pathogenesis have demonstrated that enterotoxicity can result from both whole bacteria and secreted components , indicating complex virulence mechanisms involving multiple bacterial factors. As a transmembrane protein, CPF_1836 could contribute to this process by facilitating bacterial adaptation to the host environment or participating in toxin production/secretion pathways.

What methodological approaches would best determine if CPF_1836 could serve as a therapeutic target or diagnostic marker?

To evaluate CPF_1836's potential as a therapeutic target or diagnostic marker, researchers should employ a systematic approach incorporating multiple methodologies:

When designing these studies, researchers should consider the epidemiological context of C. perfringens infections, which can involve heterogeneous bacterial populations. For example, foodborne outbreak investigations have revealed that multiple strains with different virulence profiles can be present in a single outbreak . This heterogeneity should inform the design of both therapeutic and diagnostic approaches targeting CPF_1836.

How can researchers address the challenges of studying membrane proteins like CPF_1836 in functional assays?

Membrane proteins like CPF_1836 present specific challenges for functional characterization. Researchers can address these challenges through specialized approaches:

• Solubilization and Stabilization Strategies:

  • Selection of appropriate detergents (e.g., DDM, LMNG, or digitonin)

  • Use of nanodiscs or lipid bilayer systems to maintain native environment

  • Amphipol stabilization for structural studies

  • Systematic buffer optimization to prevent aggregation

• Expression System Optimization:

  • Membrane protein-specific expression vectors

  • Lower expression temperature (e.g., 18-25°C) to improve folding

  • Fusion with stabilizing partners (e.g., MBP, GFP) that report on folding status

  • Codon optimization for membrane protein expression

• Functional Reconstitution Approaches:

  • Proteoliposome reconstitution for transport/channel assays

  • Supported lipid bilayers for microscopy-based studies

  • Tethered bilayer systems for electrical measurements

  • Cell-free expression directly into liposomes

• Advanced Biophysical Characterization:

  • Single-particle cryo-EM for structural determination

  • Solid-state NMR for structure in membrane environment

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Surface plasmon resonance in lipid environments for interaction studies

Researchers working with CPF_1836 should particularly note its transmembrane nature with multiple hydrophobic segments , which will necessitate careful consideration of detergent selection during purification and handling procedures. The N-terminal 10xHis-tag provided in recombinant versions offers a convenient purification handle but may need to be removed for certain functional studies to prevent interference with native protein activity.

How does CPF_1836 fit within the broader virulence factor landscape of Clostridium perfringens?

To position CPF_1836 within the broader virulence context of C. perfringens, researchers should consider:

• Comparative Expression Analysis:
  While the specific role of CPF_1836 in virulence is not fully characterized, its potential contribution can be evaluated through expression correlation with established virulence factors. C. perfringens virulence is typically associated with toxin production, with strains classified into types A-G based on the combination of major toxins produced . Researchers should examine whether CPF_1836 expression correlates with specific toxin profiles or is consistently expressed across diverse strain types.

• Functional Categorization:
  Based on transmembrane prediction and sequence analysis , CPF_1836 likely functions in membrane-associated processes rather than as a direct toxin. Within the broader virulence landscape, it may contribute to:

  • Environmental adaptation during infection

  • Nutrient acquisition in the host environment

  • Stress resistance mechanisms

  • Toxin secretion or regulation systems

• Genomic Context Analysis:
  The genomic neighborhood of CPF_1836 may provide clues to its functional role. Researchers should examine whether it resides within known pathogenicity islands or gene clusters associated with virulence, similar to approaches used in large-scale genomic analyses of C. perfringens .

• Virulence Gene Profiling:
  Modern virulence typing involves comprehensive profiling of multiple genes. Recent studies have employed real-time PCR techniques targeting up to 17 virulence factor genes to characterize foodborne outbreak strains . Integration of CPF_1836 into such typing schemes would help establish its relationship to known virulence determinants.

What experimental design would best determine the role of CPF_1836 in enterotoxemia models?

To investigate CPF_1836's potential role in enterotoxemia, researchers should design experiments that build upon established animal models while incorporating specific analyses for this protein:

• Modified Enterotoxemia Model Design:

  • Base design on validated goat enterotoxemia models

  • Include experimental groups: (a) wild-type C. perfringens, (b) CPF_1836 knockout strain, (c) complemented strain, (d) negative controls

  • Administer via intraduodenal route following established protocols

  • Monitor for clinical signs including CNS symptoms and diarrhea

• Comparative Group Analysis:

  Experimental Group	Expected Outcome if CPF_1836 is Involved	Expected Outcome if CPF_1836 is Not Involved
  Wild-type bacteria	Full disease manifestation	Full disease manifestation
  CPF_1836 knockout	Attenuated symptoms	No change in disease progression
  Complemented strain	Restored virulence	No change from wild-type
  Purified CPF_1836 protein	Specific pathological changes	No significant effects
  Control (culture medium)	No disease	No disease

• Comprehensive Assessment Metrics:

  • Clinical scoring systems for neurological signs and diarrhea

  • Histopathological evaluation focusing on lung edema, colitis, and cerebral changes

  • Immunohistochemistry to track bacterial colonization and CPF_1836 localization

  • Transcriptomic analysis of host response to different bacterial strains

  • Measurement of inflammatory markers and tissue damage indicators

• Mechanistic Follow-up Studies:

  • Ex vivo intestinal loop models to assess direct effects on epithelium

  • Bacterial transcriptomics to identify compensatory changes in CPF_1836 mutants

  • Metabolomic profiling to identify altered bacterial or host metabolites

This experimental design builds on the established finding that in enterotoxemia models, both whole cultures and filtered supernatants can induce disease, suggesting complex pathogenicity mechanisms involving both cellular and secreted components .

How can researchers integrate CPF_1836 studies with current understanding of C. perfringens genomic diversity?

Integrating CPF_1836 research with current understanding of C. perfringens genomic diversity requires multifaceted approaches:

• Strain Diversity Sampling:

  • Include diverse isolates representing different:

    • Phylogenetic clades identified through SNP-based analyses

    • Toxinotypes (Types A-G)

    • Source environments (clinical, food, environmental)

    • Geographic origins

  • This approach aligns with comprehensive genomic studies that have revealed significant strain heterogeneity even within outbreaks

• Comparative Genomic Analysis:

  • Sequence the CPF_1836 gene and flanking regions across strain collections

  • Identify single nucleotide polymorphisms (SNPs) and structural variants

  • Construct gene-specific phylogenetic trees to compare with whole-genome phylogenies

  • Analyze selection pressures (dN/dS ratios) to identify functional constraints

• Association Studies:

  • Correlate CPF_1836 sequence variants with:

    • Virulence phenotypes in experimental models

    • Clinical disease associations

    • Presence/absence of other virulence factors

    • Chromosomal versus plasmid-borne toxin genes

• Multi-Omics Integration:

  • Combine genomic data with:

    • Transcriptomic profiles under different conditions

    • Proteomic analyses of membrane fractions

    • Functional characterization of variant proteins

    • Metabolomic signatures of different strains

This integrative approach would build upon findings from large-scale genomic analyses of C. perfringens that have identified distinct clades differentiating chromosomal cpe-positive strains from cpe-negative and plasmid-borne cpe strains , providing a framework for understanding how CPF_1836 variants might correlate with established genomic diversity patterns.

What are common technical challenges when working with recombinant CPF_1836 and how can they be addressed?

Researchers working with recombinant CPF_1836 may encounter several technical challenges inherent to transmembrane proteins. Here are common issues and their solutions:

• Protein Aggregation and Precipitation:

  • Challenge: Transmembrane proteins often aggregate during purification or storage

  • Solutions:

    • Optimize detergent type and concentration (try LMNG, DDM, or digitonin)

    • Include glycerol (10-20%) in storage buffers

    • Store at higher concentrations with carrier proteins when appropriate

    • Use trehalose (as in commercial preparations) to enhance stability

    • Consider nanodiscs or amphipol stabilization for functional studies

• Low Expression Yields:

  • Challenge: Membrane proteins typically express at lower levels than soluble proteins

  • Solutions:

    • Reduce expression temperature to 16-18°C

    • Use specialized expression strains (C41/C43, Lemo21)

    • Optimize codon usage for E. coli

    • Consider fusion partners that enhance expression (MBP, SUMO)

    • Scale up culture volumes to compensate for lower per-cell yield

• Protein Activity Loss:

  • Challenge: Native function may be compromised during purification

  • Solutions:

    • Minimize time between cell lysis and final purification

    • Include protease inhibitors throughout purification

    • Maintain constant low temperature during handling

    • Consider activity assays at each purification step

    • Re-validate activity after freeze-thaw cycles

• Tag Interference:

  • Challenge: The N-terminal 10xHis-tag may affect function

  • Solutions:

    • Include tag-only controls in functional assays

    • Consider tag removal with specific proteases

    • Compare N-terminal and C-terminal tagged versions

    • Use cleavable tags when function is compromised

These approaches are particularly important for CPF_1836 given its transmembrane nature with multiple hydrophobic regions identified in its sequence , which make it prone to the challenges typical of membrane proteins.

How can researchers validate antibodies and other detection tools for CPF_1836 studies?

Thorough validation of detection tools is essential for reliable CPF_1836 research. The following comprehensive validation strategy is recommended:

• Antibody Validation Protocol:

  Validation Approach	Method	Success Criteria
  Specificity Testing	Western blot against recombinant protein	Single band at expected MW (~20 kDa plus tag)
  	Immunoblot against CPF_1836 knockout	Absence of signal compared to wild-type
  	Competition with purified antigen	Signal reduction proportional to competitor
  Sensitivity Assessment	Limit of detection determination	Detection of ≤10 ng purified protein
  	Signal-to-noise ratio calculation	Ratio >10:1 at working concentration
  Cross-reactivity Testing	Testing against related UPF0397 family proteins	<10% signal compared to CPF_1836
  	Screening against host tissue lysates	No significant non-specific binding
  Application Validation	Immunofluorescence microscopy	Membrane localization pattern
  	Flow cytometry	Positive signal in intact bacteria
  	Immunoprecipitation efficiency	>50% target protein recovery

• PCR-based Detection Tools:

  • Design primers spanning unique regions of CPF_1836

  • Validate specificity across C. perfringens strains

  • Determine limit of detection using serial dilutions

  • Compare sensitivity between conventional and real-time PCR

  • Include internal amplification controls

• Mass Spectrometry Methods:

  • Identify unique peptide signatures for CPF_1836

  • Develop multiple reaction monitoring (MRM) assays

  • Establish standard curves with purified protein

  • Validate extraction methods from complex matrices

  • Determine interference profiles from biological samples

• Reporter Systems:

  • Construct fluorescent protein fusions (considering topology constraints)

  • Validate expression and localization in C. perfringens

  • Compare fusion protein function to native protein

  • Develop inducible systems for temporal studies

These validation approaches should consider the transmembrane nature of CPF_1836 , which may affect epitope accessibility in different applications and require specialized extraction procedures for detection from bacterial cultures.

What considerations are important when designing gene knockout or mutation studies for CPF_1836?

When designing genetic manipulation studies for CPF_1836, researchers should address several critical considerations:

• Genetic Manipulation Strategy Selection:

  • Allelic Exchange: Preferred for precise deletions or substitutions

  • Group II Intron Systems (e.g., ClosTron): Effective for insertional inactivation

  • CRISPR-Cas9 Approaches: For precise genome editing when transformation efficiency is sufficient

  • Antisense RNA: For conditional knockdown if knockout is lethal

• Essential Gene Considerations:

  • Generate conditional mutants if initial attempts at knockout fail

  • Consider domain-specific deletions rather than whole-gene knockout

  • Implement depletion strategies with inducible promoters

  • Prepare complementation constructs before attempting knockout

• Membrane Protein-Specific Challenges:

  • Account for potential polar effects on flanking genes

  • Consider topology disruption in partial deletions

  • Design mutations that maintain membrane integrity

  • Include membrane protein stabilizing elements in complementation constructs

• Validation Requirements:

  • RT-qPCR to confirm transcript elimination

  • Western blot to verify protein absence

  • Genome sequencing to confirm precise modification

  • Phenotypic rescue with complementation

  • Controls for unintended off-target effects

• Experimental Design Considerations:

  • Include multiple independent mutant clones

  • Careful selection of control strains

  • Comprehensive phenotypic characterization

  • Consider selecting knockout sites based on strain variability information from genomic studies

When interpreting results from these genetic studies, researchers should consider the broader genomic context. C. perfringens strains exhibit significant genomic diversity, with distinct clades identified through SNP-based analyses . This diversity may influence the phenotypic consequences of CPF_1836 manipulation in different strain backgrounds.

What emerging technologies could advance our understanding of CPF_1836 function?

Several cutting-edge technologies offer promising approaches to elucidate CPF_1836 function:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle analysis for high-resolution structure determination

  • In situ structural studies within native membrane environments

  • Visualization of protein-protein interactions in membrane complexes

  • Time-resolved cryo-EM to capture conformational dynamics

• Advanced Genetic Manipulation Technologies:

  • CRISPR interference (CRISPRi) for tunable gene repression

  • Base editing for precise amino acid substitutions without double-strand breaks

  • In vivo transposon mutagenesis with deep sequencing (TnSeq) to map functional domains

  • Inducible degron systems for temporal control of protein levels

• Single-Cell and Spatial Technologies:

  • Single-cell transcriptomics to capture heterogeneity in bacterial populations

  • Spatial transcriptomics to map gene expression in infection microenvironments

  • Mass cytometry for high-dimensional protein analysis at single-cell resolution

  • Super-resolution microscopy for nanoscale localization and dynamics

• Artificial Intelligence Integration:

  • Deep learning for structure prediction and functional annotation

  • Machine learning analysis of phenotypic screens

  • Network-based approaches to predict protein-protein interactions

  • Automated literature mining to generate functional hypotheses

These emerging technologies could be particularly valuable for understanding CPF_1836 in the context of C. perfringens heterogeneity, where studies have revealed complex relationships between genomic profiles and virulence characteristics . For instance, advanced genomic approaches have identified unexpected links between different outbreaks associated with closely related strains , suggesting that similar approaches could reveal important functional aspects of CPF_1836.

How might comparative studies across different Clostridium species inform our understanding of CPF_1836?

Comparative studies across Clostridium species can provide valuable evolutionary and functional insights into CPF_1836:

• Evolutionary Trajectory Analysis:

  • Reconstruct phylogenetic history of UPF0397 family proteins

  • Identify conserved versus variable regions across species

  • Map sequence divergence to structural features

  • Correlate evolutionary patterns with pathogenicity profiles

• Structure-Function Relationship Mapping:

  • Compare predicted structures across homologs

  • Identify functionally critical residues through conservation analysis

  • Correlate structural variations with phenotypic differences

  • Design chimeric proteins to test domain-specific functions

• Niche Adaptation Correlation:

  • Analyze UPF0397 protein variants in relation to:

    • Host range differences between Clostridium species

    • Environmental persistence capabilities

    • Growth requirements and metabolic capabilities

    • Stress response mechanisms

• Horizontal Gene Transfer Assessment:

  • Identify potential interspecies transfer events

  • Evaluate selective pressures in different genomic contexts

  • Analyze flanking mobile genetic elements

  • Compare gene organization across species

This comparative approach builds on methodologies used in large-scale genomic analyses of C. perfringens, where whole genome sequence phylogenomic reconstruction has revealed important clade structures corresponding to virulence characteristics . Extending such approaches to examine CPF_1836 homologs across Clostridium species could provide evolutionary context for its function in C. perfringens.

What multidisciplinary approaches would most effectively advance CPF_1836 research in the next five years?

To substantially advance CPF_1836 research over the next five years, integrated multidisciplinary approaches combining complementary methodologies will be most effective:

• Integrated Structural Biology Pipeline:

  • AlphaFold structure prediction for initial modeling

  • Cryo-EM for experimental structure determination

  • Molecular dynamics simulations in membrane environments

  • Structure-guided functional hypothesis generation

  • Structure-based virtual screening for small molecule probes

• Systems Biology Framework:

  • Multi-omics profiling (transcriptomics, proteomics, metabolomics)

  • Network analysis to position CPF_1836 in cellular pathways

  • Flux balance analysis to identify metabolic impacts

  • Perturbation studies with temporal profiling

  • Integration with existing C. perfringens datasets

• Translational Research Collaboration:

  • Animal model development with veterinary specialists

  • Clinical strain collection and characterization

  • Diagnostic test development and validation

  • Therapeutic targeting feasibility assessment

  • One Health approach connecting environmental, animal, and human aspects

• Advanced Computing Integration:

  • Machine learning for pattern recognition in complex datasets

  • Quantum computing applications for molecular modeling

  • Cloud-based collaborative platforms for data sharing

  • Automated literature mining for hypothesis generation

  • Predictive modeling of protein-protein interactions

This multidisciplinary strategy aligns with current trends in C. perfringens research, where studies have demonstrated the value of integrating genomic analyses with phenotypic characterization and epidemiological data . Such holistic approaches have revealed unexpected links between seemingly distinct outbreaks and could similarly uncover important aspects of CPF_1836 function in different contexts.