Recombinant Corynebacterium jeikeium UPF0233 membrane protein jk0035 (jk0035)

What is Corynebacterium jeikeium UPF0233 membrane protein jk0035?

Corynebacterium jeikeium UPF0233 membrane protein jk0035 (UniProt ID: Q4JYC3) is a 90-amino acid membrane protein also known as CrgA (Cell division protein CrgA). The full amino acid sequence is: MPKSKVNSAEENYSSSSSADRRTPVKLNSSGTPRWYIVLMLALMLLGLAWLVVNYIAGPE IPFMRDLNAWNYLIGFALLIVGLLMTMGWK . This protein belongs to the UPF0233 family of membrane proteins and is found in Corynebacterium jeikeium, a bacterium associated with serious infections, particularly in immunocompromised patients. C. jeikeium is notably linked to bacteremia in patients with hematologic malignancies, with a 30% mortality rate in true bacteremia cases .

What are the optimal methods for reconstituting recombinant jk0035 protein?

The optimal reconstitution method for recombinant jk0035 protein involves:

• Brief centrifugation of the vial prior to opening to bring contents to the bottom

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol (5-50% final concentration) as a cryoprotectant

• Aliquoting for long-term storage to avoid freeze-thaw cycles

For experimental work, researchers should consider reconstituting the protein in a buffer system that maintains physiological pH (typically Tris/PBS-based buffer, pH 8.0), as the protein is typically lyophilized in this buffer with 6% trehalose as a stabilizing agent .

What are the structural characteristics of jk0035 membrane protein?

The jk0035 protein has several notable structural characteristics:

• A full length of 90 amino acids

• Hydrophobic regions consistent with transmembrane domains (evident from the amino acid sequence with stretches of hydrophobic amino acids: WYIVLMLALMLLGLAWLVVNY and NYWNYLIGFALLIVGLLMTMGWK)

• Likely orientation across the bacterial cell membrane based on its sequence characteristics

The membrane integration of this protein makes structural studies challenging, similar to other membrane proteins that require specialized techniques such as cryo-EM or X-ray crystallography following stabilization in lipid nanoparticles or detergent micelles .

What methodologies are most effective for structural analysis of jk0035 membrane protein?

Based on current advances in membrane protein research, the following methodologies are most effective for structural analysis of jk0035:

For jk0035 specifically, direct extraction into lipid Salipro nanoparticles from expression systems might be particularly effective, allowing for subsequent structure-function analysis using SPR and cryo-EM . This approach has been successful with other challenging membrane proteins and could be adapted for jk0035.

How might the function of jk0035 relate to C. jeikeium pathogenesis in clinical settings?

The function of jk0035, also known as cell division protein CrgA, may have significant implications for C. jeikeium pathogenesis based on clinical observations:

• C. jeikeium causes true bacteremia at significantly higher rates (71%) than other Corynebacterium species (except C. striatum)

• C. jeikeium bacteremia is particularly common in patients with hematologic malignancies (64% of cases) and neutropenia

• The 90-day mortality rate for C. jeikeium bacteremia is approximately 30%

Given that jk0035/CrgA is involved in cell division, it may contribute to pathogenesis through:

• Regulation of bacterial replication during infection

• Adaptation to the host environment during bacteremia

• Potential interactions with host immune cells or proteins

• Possible role in antimicrobial resistance mechanisms

Experimental approaches to investigate this relationship would include:

• Construction of jk0035 knockout mutants and assessment of virulence in vitro and in vivo

• Transcriptomic analysis comparing jk0035 expression levels in clinical isolates versus laboratory strains

• Protein-protein interaction studies to identify host targets of jk0035 during infection

What challenges exist in expressing and purifying functional jk0035 protein for research?

Expressing and purifying functional jk0035 presents several significant challenges:

• Membrane protein solubility issues:

  • Hydrophobic transmembrane domains can cause aggregation

  • Selection of appropriate detergents is critical for extraction from membranes

  • Maintaining native-like conformation during purification requires careful optimization

• Expression system considerations:

  • E. coli systems (commonly used) may not provide proper folding environment

  • Codon optimization may be necessary for efficient expression

  • Fusion tags (like the His-tag) must be positioned to avoid interfering with protein function

• Purification strategy optimization:

  • Metal affinity chromatography using His-tag is effective but requires optimization of imidazole concentrations

  • Size exclusion chromatography helps separate monomeric from aggregated protein

  • Quality control through SDS-PAGE and Western blotting is essential to confirm purity (>90% is typically achievable)

• Functional assessment challenges:

  • Lack of established activity assays for jk0035

  • Need for reconstitution into membrane-like environments to assess function

  • Limited knowledge of binding partners or substrates

Researchers have overcome similar challenges with other membrane proteins by using novel extraction methods that directly incorporate the protein into lipid nanoparticles, preserving functionality for downstream analyses .

How can researchers design experiments to elucidate the role of jk0035 in C. jeikeium clinical isolates?

A comprehensive experimental design to elucidate the role of jk0035 in clinical isolates would include:

• Clinical isolate characterization:

  • Collection of C. jeikeium isolates from bacteremia cases, particularly from patients with hematologic malignancies

  • Genome sequencing to identify polymorphisms in the jk0035 gene

  • Transcriptomic analysis to determine jk0035 expression levels in different clinical contexts

• Genetic manipulation experiments:

  • Construction of jk0035 knockout mutants using CRISPR-Cas9 or traditional homologous recombination

  • Complementation studies to confirm phenotypes

  • Site-directed mutagenesis to assess the impact of clinically observed polymorphisms

• Phenotypic characterization:

  • Growth curves under various conditions (temperature, pH, nutrient limitation)

  • Cell morphology and division pattern analysis using electron microscopy

  • Antibiotic susceptibility testing compared to wild-type strains

  • Biofilm formation capacity assessment

• Host-pathogen interaction studies:

  • Adhesion and invasion assays using relevant human cell lines

  • Neutrophil survival assays (particularly relevant given association with neutropenic patients)

  • Serum resistance testing

  • In vivo infection models (if ethically approved)

• Proteomic approaches:

  • Identification of jk0035 interaction partners using pull-down assays

  • Phosphoproteomics to identify signaling pathways affected by jk0035

  • Comparative proteomics between wild-type and jk0035 mutants

This multifaceted approach would provide comprehensive insights into the role of jk0035 in clinically relevant contexts, particularly its potential contribution to the high mortality associated with C. jeikeium bacteremia in patients with hematologic malignancies .

What methodological approaches can be applied to study membrane protein dynamics of jk0035?

Understanding membrane protein dynamics requires specialized methodological approaches:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Provides information on protein flexibility and conformational changes

  • Can identify regions involved in ligand binding or protein-protein interactions

  • Requires careful optimization for membrane proteins like jk0035

• Single-molecule Förster resonance energy transfer (smFRET):

  • Measures distances between fluorescently labeled residues

  • Captures conformational dynamics in real-time

  • Requires strategic placement of fluorophores based on structural predictions

• Molecular dynamics (MD) simulations:

  • Predicts protein behavior in membrane environments

  • Can model conformational changes over nanosecond to microsecond timescales

  • Requires validation with experimental data

• Site-directed spin labeling with electron paramagnetic resonance (SDSL-EPR):

  • Measures distances between spin-labeled residues

  • Works well in membrane environments

  • Provides information on conformational distributions

Similar approaches have been successfully applied to study other bacterial membrane proteins, such as AVPR2, where disease-causing mutations were found to alter structural dynamics . For jk0035, these techniques could reveal how the protein functions in cell division and potentially identify targets for therapeutic intervention against C. jeikeium infections.

What controls should be included when studying recombinant jk0035 protein function?

When designing experiments to study jk0035 function, the following controls are essential:

• Negative controls:

  • Heat-denatured jk0035 protein to confirm activity is protein-specific

  • Empty vector-expressed product purified identically to control for host cell contaminants

  • Buffer-only controls for all functional assays

• Positive controls:

  • Well-characterized membrane protein with similar size/structure

  • Native (non-recombinant) jk0035 if available

  • Recombinant protein with known activity from the same expression system

• Specificity controls:

  • Structurally similar but functionally distinct membrane proteins

  • Antibody pre-absorption controls for immunological detection

  • Competitive binding assays with unlabeled protein

• Expression system controls:

  • Comparison of E. coli-expressed protein with protein expressed in other systems

  • Tag-free protein compared to His-tagged version to assess tag interference

  • Multiple purification lots to ensure reproducibility

Proper controls ensure that observed effects are specifically attributable to jk0035 function rather than experimental artifacts or contaminants.

How can researchers accurately assess the purity and functionality of recombinant jk0035 preparations?

Accurate assessment of jk0035 purity and functionality requires multiple complementary techniques:

For membrane proteins like jk0035, additional considerations include:

• Detergent screening to identify optimal conditions for stability

• Reconstitution into proteoliposomes to verify membrane integration

• Assessment of orientation in membranes through protease protection assays

These multifaceted approaches ensure that subsequent functional studies are performed with properly folded, pure, and active protein preparations.

How might understanding jk0035 contribute to novel therapeutic approaches for C. jeikeium infections?

Understanding jk0035 structure and function could contribute to novel therapeutic approaches in several ways:

• Drug target potential:

  • As a membrane protein involved in cell division, jk0035 represents a potential antibiotic target

  • Structural information could enable structure-based drug design

  • Inhibiting jk0035 function might specifically target C. jeikeium without affecting beneficial microbiota

• Relevance to high-risk populations:

  • C. jeikeium predominantly affects patients with hematologic malignancies (64% of cases)

  • Targeted therapies could benefit this vulnerable population

  • Potential for prophylactic treatments in high-risk neutropenic patients

• Addressing antibiotic resistance:

  • C. jeikeium isolates show resistance to multiple antibiotics

  • Novel targets like jk0035 could overcome existing resistance mechanisms

  • Combination therapies targeting jk0035 and other pathways might prevent resistance development

• Diagnostic applications:

  • Recombinant jk0035 could be used to develop specific antibodies for rapid C. jeikeium detection

  • Expression patterns of jk0035 might serve as biomarkers for virulence or treatment response

  • Understanding jk0035 function might explain the high rate (71%) of true bacteremia caused by C. jeikeium compared to other Corynebacterium species

Research into membrane proteins has already shown promise for developing new therapeutics for other diseases, as demonstrated by work on AVPR2 for diabetes insipidus , suggesting similar potential for jk0035-targeted approaches.

What bioinformatic approaches can predict potential interaction partners of jk0035?

Several bioinformatic approaches can be applied to predict potential interaction partners of jk0035:

• Homology-based prediction:

  • Identification of interaction partners of homologous proteins in related species

  • Application of interolog mapping (transfer of protein interactions across species)

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

• Structure-based prediction:

  • Molecular docking simulations with potential partners

  • Interface prediction using surface characteristics

  • Coarse-grained molecular dynamics to assess binding energetics

• Genomic context methods:

  • Operon analysis to identify functionally related genes

  • Gene neighborhood conservation across species

  • Gene fusion events indicating functional relationships

• Network-based approaches:

  • Co-expression network analysis using transcriptomic data

  • Protein-protein interaction network integration

  • Text mining of scientific literature for reported associations

• Machine learning approaches:

  • Integration of multiple data types for partner prediction

  • Feature-based classification of potential interactions

  • Deep learning methods for interaction prediction from sequence data

These computational predictions would require experimental validation through techniques such as co-immunoprecipitation, bacterial two-hybrid systems, or direct binding assays using the recombinant protein.

How can researchers overcome the challenges of expressing membrane proteins like jk0035 in heterologous systems?

Expressing membrane proteins like jk0035 in heterologous systems presents significant challenges that can be addressed through several strategies:

• Expression system optimization:

  • E. coli strains specifically designed for membrane protein expression (C41, C43, Lemo21)

  • Use of weak promoters to prevent overwhelming the membrane insertion machinery

  • Low-temperature induction to slow protein production and improve folding

  • Codon optimization for the expression host

• Fusion partner approaches:

  • N-terminal fusions with highly soluble proteins (MBP, SUMO, Trx)

  • Addition of signal sequences for proper membrane targeting

  • Strategic placement of affinity tags to avoid interference with membrane domains

• Media and growth condition optimization:

  • Supplementation with specific lipids similar to native C. jeikeium membrane

  • Addition of chemical chaperones to improve folding

  • Osmotic stress adaptation to enhance membrane protein yield

  • Use of defined minimal media to control growth rate

• Alternative expression systems:

  • Cell-free expression systems with supplied lipids or detergents

  • Yeast expression systems for eukaryotic-like quality control

  • Insect cell expression for complex membrane proteins

• Direct extraction methods:

  • Novel approaches using lipid Salipro nanoparticles to directly extract membrane proteins from cells

  • Nanodiscs for native-like membrane environment

  • Amphipols as detergent alternatives

These approaches have been successfully applied to other challenging membrane proteins and could be adapted for jk0035 expression and purification, potentially improving yield and maintaining native conformation and function.

What are the most reliable methods for assessing membrane integration and orientation of recombinant jk0035?

Assessing membrane integration and orientation of recombinant jk0035 requires specialized techniques:

• Protease protection assays:

  • Treatment of proteoliposomes with proteases that cannot cross membranes

  • Analysis of protected fragments by mass spectrometry

  • Comparison with computational topology predictions

• Fluorescence-based approaches:

  • Site-specific labeling of predicted extramembranous regions

  • Quenching experiments with membrane-impermeable quenchers

  • FRET measurements between strategically placed fluorophores

• Antibody accessibility studies:

  • Generation of antibodies against specific domains

  • Differential accessibility in intact versus permeabilized membranes

  • Immunogold electron microscopy for direct visualization

• Chemical modification approaches:

  • Selective labeling of accessible cysteine residues

  • Mass spectrometry to identify modified sites

  • Comparison with computational topology models

• Biophysical techniques:

  • Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR)

  • Oriented circular dichroism (OCD)

  • Neutron reflectometry for membrane positioning

These methodologies provide complementary information about membrane integration and orientation, crucial for understanding jk0035 function and for the development of targeted therapeutics against C. jeikeium infections.