Recombinant Citrobacter koseri UPF0060 membrane protein CKO_01576 (CKO_01576)

What is the structural organization of CKO_01576 protein?

CKO_01576 is classified as a UPF0060 family membrane protein in Citrobacter koseri with a full length of 108 amino acids . As a recombinant protein, it is typically expressed with a His-tag in E. coli expression systems to facilitate purification . While the complete three-dimensional structure has not been fully resolved, membrane proteins typically contain transmembrane domains that anchor them within the bacterial cell membrane.

The protein belongs to the broader category of uncharacterized protein families (UPF), specifically UPF0060, which indicates proteins grouped based on sequence similarity without fully elucidated functions. Membrane localization suggests potential roles in:

• Molecular transport

• Signal transduction

• Cell wall maintenance

• Antibiotic resistance mechanisms

For preliminary structural analysis, researchers should consider multiple computational prediction tools to generate consensus models before proceeding to experimental validation.

How does CKO_01576 compare with other membrane proteins in C. koseri?

When analyzing CKO_01576 within the context of other C. koseri membrane proteins, researchers should consider both sequence homology and potential functional relationships. C. koseri is a gram-negative rod associated with infections in immunocompromised individuals, particularly urinary tract infections . Membrane proteins in such pathogens often contribute to virulence and antibiotic resistance mechanisms.

Comparative analysis should include:

Understanding these relationships may provide initial functional hypotheses about CKO_01576's role in C. koseri biology and potentially in its pathogenicity.

What are the optimal conditions for recombinant expression of CKO_01576?

Successful expression of membrane proteins like CKO_01576 requires careful optimization of multiple parameters. Based on established protocols for membrane protein expression, researchers should consider:

Expression system selection: E. coli has been successfully used for CKO_01576 expression , but alternative systems should be considered if facing solubility challenges:

For E. coli expression, consider these methodological optimizations:

• Reduce expression temperature to 18-25°C after induction

• Use lower IPTG concentrations (0.1-0.5 mM) for induction

• Employ specialized media formulations with osmolytes

• Co-express with molecular chaperones if misfolding occurs

• Consider fusion partners that enhance membrane protein solubility

These optimization strategies can significantly improve the yield and quality of recombinant CKO_01576, enhancing downstream experimental applications.

What purification strategies yield the highest purity and activity for CKO_01576?

Purification of His-tagged CKO_01576 should follow a multi-step approach to ensure both purity and retention of native conformation:

• Membrane extraction: The critical first step involves careful solubilization of the membrane fraction containing CKO_01576:

  • Screen multiple detergents (DDM, LDAO, Triton X-100) at varying concentrations

  • Optimize buffer conditions (pH, ionic strength, glycerol content)

  • Consider using detergent:protein ratios between 2:1 and 5:1

• Immobilized metal affinity chromatography (IMAC):

  • Utilize the His-tag for initial capture

  • Include low concentrations of detergent in all purification buffers

  • Consider gradient elution with imidazole rather than step elution

  • Monitor protein quality at each step via SDS-PAGE and Western blotting

• Secondary purification methods:

  • Size exclusion chromatography to isolate monomeric protein

  • Ion exchange chromatography if additional purity is required

  • Affinity tag removal if desired using TEV or similar proteases

The purified protein should be characterized for homogeneity, stability, and activity before proceeding to functional studies. Membrane protein purification typically yields lower amounts compared to soluble proteins, so optimizing each step is crucial for experimental success.

How can the functional role of CKO_01576 be experimentally determined?

Given the uncharacterized nature of UPF0060 family proteins, a systematic approach to functional elucidation is recommended:

Computational approaches as starting points:

• Sequence-based function prediction using multiple algorithms

• Structural modeling to identify potential binding sites or catalytic regions

• Analysis of genomic context and gene neighborhood in C. koseri

Experimental validation strategies:

• Loss-of-function studies:

  • Generate knockout or knockdown mutants in C. koseri

  • Phenotypic characterization (growth curves, stress resistance)

  • Transcriptomic/proteomic profiling of mutants versus wild-type

• Protein interaction studies:

  • Pull-down assays using purified His-tagged CKO_01576

  • Bacterial two-hybrid screening

  • Cross-linking mass spectrometry to identify interaction partners

• Localization studies:

  • Immunofluorescence microscopy with anti-His antibodies

  • GFP fusion protein localization if tolerated

  • Subcellular fractionation and Western blotting

The combination of computational predictions and experimental validation offers the most comprehensive approach to determining CKO_01576 function in C. koseri biology.

What role might CKO_01576 play in C. koseri pathogenesis?

C. koseri is associated with infections in immunocompromised individuals, particularly urinary tract infections . Membrane proteins often contribute to pathogenesis through various mechanisms. While specific information about CKO_01576's role is limited, potential contributions could include:

Potential pathogenic roles to investigate:

• Adhesion to host tissues:

  • Binding assays with relevant host cell types

  • Competitive inhibition studies with recombinant protein

  • Comparison with known adhesins using structural homology

• Antibiotic resistance:

  • MIC determinations in wild-type versus knockout strains

  • Transport assays for potential efflux activity

  • Membrane permeability assessments

• Immune evasion:

  • Interaction studies with host immune components

  • Survival assays in presence of antimicrobial peptides

  • Inflammatory response measurements in cell culture models

Given that C. koseri has acquired resistance to current antibiotics , investigating CKO_01576's potential contribution to resistance mechanisms could provide valuable insights for therapeutic development.

How can CKO_01576 be evaluated as a potential vaccine target for C. koseri infections?

Recent research has employed subtractive proteomics to identify potential vaccine targets against C. koseri . While CKO_01576 wasn't specifically identified in that study, the methodology provides a framework for evaluating its potential as a vaccine candidate:

Antigenicity assessment workflow:

• In silico prediction:

  • Epitope mapping using B-cell and T-cell epitope prediction tools

  • Antigenicity scoring using multiple algorithms

  • Conservation analysis across C. koseri strains

• Experimental validation:

  • Expression and purification of recombinant CKO_01576

  • Immunogenicity testing in animal models

  • Antibody generation and functional characterization

• Protection studies:

  • Challenge experiments in appropriate animal models

  • Antibody neutralization assays

  • Cross-protection assessment against different strains

The successful vaccine development described in the literature identified DP-3-O-acyl-N-acetylglucosamine deacetylase and Arabinose 5-phosphate isomerase as potential targets , providing comparative benchmarks for CKO_01576 evaluation.

How can structural studies of CKO_01576 be approached considering its membrane protein nature?

Structural characterization of membrane proteins presents unique challenges but is crucial for understanding function. For CKO_01576, a multi-technique approach is recommended:

Progressive structural characterization strategy:

Since CKO_01576 is relatively small for a membrane protein (108 amino acids) , it may be amenable to solution NMR approaches if suitable conditions can be identified. Alternatively, X-ray crystallography using lipidic cubic phase methods could be pursued.

How should RNA-sequencing data be analyzed to understand CKO_01576 expression patterns?

RNA-sequencing can provide valuable insights into the expression patterns of CKO_01576 under various conditions. Based on methodologies described for gene expression analysis in the search results , researchers should:

• Data preprocessing and normalization:

  • Apply MAS5 normalization followed by scaling normalization to standardize mean expression

  • Implement quality control measures to assess background intensity, noise levels, and percentage of present calls

  • Filter data to ensure signal is above background (>200, twice the background intensity)

• Differential expression analysis:

  • Use Cox proportional hazards regression analysis for time-course experiments

  • Consider all available cutoff values between lower and upper quartiles of expression

  • Apply Benjamini-Hochberg method to compute FDR and correct for multiple testing

• Contextual interpretation:

  • Analyze co-expressed genes for potential functional relationships

  • Examine expression in different growth conditions and infection models

  • Compare with expression of genes in the same genomic neighborhood

This structured approach allows for robust analysis of CKO_01576 expression patterns, potentially revealing conditions where the protein plays critical roles in C. koseri biology.

What integrative approaches can be used to predict CKO_01576 function from diverse datasets?

Modern functional prediction requires integration of multiple data types. Researchers investigating CKO_01576 should consider:

Multi-omics integration strategy:

• Genomic context analysis:

  • Examine conservation of gene neighborhood across related species

  • Identify potential operons containing CKO_01576

  • Analyze promoter regions for regulatory elements

• Transcriptomic correlation networks:

  • Build co-expression networks from multiple conditions

  • Identify gene clusters with similar expression patterns

  • Apply guilt-by-association principles for functional inference

• Proteomic interaction mapping:

  • Use purified His-tagged CKO_01576 for pull-down experiments

  • Analyze resulting protein complexes by mass spectrometry

  • Construct interaction networks and identify functional modules

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and CKO_01576 mutants

  • Identify altered metabolic pathways

  • Correlate with phenotypic changes

• Machine learning integration:

  • Develop predictive models combining multiple data types

  • Validate predictions experimentally

  • Refine models iteratively with new data

This integrated approach mirrors successful strategies described for identifying gene functions in complex systems and provides the most comprehensive path to understanding CKO_01576's role in C. koseri.

How can expression and solubility issues with recombinant CKO_01576 be resolved?

Membrane proteins like CKO_01576 frequently present expression and solubility challenges. When facing such issues, consider this systematic troubleshooting approach:

Expression optimization:

• Vector design modifications:

  • Test different promoter strengths (T7, tac, ara)

  • Optimize codon usage for expression host

  • Include solubility-enhancing fusion partners (MBP, SUMO)

• Host strain selection:

  • Try C41/C43 E. coli strains specialized for membrane proteins

  • Consider Lemo21(DE3) for tunable expression

  • Evaluate Rosetta strains if rare codons are present

• Induction protocol adjustments:

  • Reduce temperature to 18-20°C during induction

  • Lower inducer concentration significantly

  • Extend expression time (overnight or longer)

Solubilization strategies:

• Detergent screening matrix:

• Alternative solubilization approaches:

  • Amphipols for increased stability post-purification

  • Nanodiscs for functional studies in lipid environment

  • Cell-free expression directly into liposomes

Systematically testing these variables while monitoring protein quality through methods like size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) will help optimize conditions for successful CKO_01576 production.

How can contradictory results in CKO_01576 functional studies be reconciled?

When facing contradictory results in functional studies of CKO_01576, researchers should implement a structured approach to reconciliation:

• Methodological evaluation:

  • Compare experimental conditions in detail across studies

  • Assess protein quality and verification methods used

  • Examine statistical approaches and significance thresholds

• Biological context considerations:

  • Evaluate strain differences in C. koseri isolates

  • Consider growth conditions and physiological states

  • Examine potential redundancy with other proteins

• Integrated validation approach:

  • Design experiments that combine multiple detection methods

  • Implement orthogonal functional assays

  • Use complementation studies to confirm specificity

• Collaborative resolution strategy:

  • Establish common protocols between laboratories

  • Exchange materials (strains, plasmids, antibodies)

  • Perform blinded replication studies

This systematic approach to reconciling contradictory results will strengthen the reliability of functional characterizations of CKO_01576 and advance understanding of its biological role.