Recombinant ESX-2 secretion system protein eccB2 (eccB2)

What is the ESX-2 secretion system and what is the role of EccB2 within it?

The ESX-2 secretion system is one of five Type VII secretion systems (T7SS) found in mycobacteria. Based on structural and functional studies of homologous ESX systems, EccB2 serves as a critical structural component of the membrane complex. In characterized ESX systems, EccB proteins contain a periplasmic domain that likely anchors the secretion apparatus to the cell wall through peptidoglycan interaction .

Methodologically, to investigate EccB2's specific function, researchers should employ:

• Gene deletion studies to assess phenotypic changes

• Protein-protein interaction assays (pull-down experiments, bacterial two-hybrid)

• Fluorescently-tagged protein localization studies

• Complementation experiments in eccB2 knockout strains

• Structural analysis via cryo-EM or X-ray crystallography

These approaches have revealed that in other ESX systems, EccB proteins form part of a hexameric membrane complex with other Ecc proteins, creating a secretion channel that spans the mycobacterial cell envelope .

How does EccB2 compare structurally with other EccB proteins in different ESX systems?

Structural comparison of EccB2 with other EccB proteins reveals both conserved features and system-specific differences:

For experimental characterization, researchers should:

• Perform multiple sequence alignments using tools like Clustal Omega

• Use AlphaFold or RoseTTAFold to generate structural models

• Compare with experimentally determined structures of other EccB proteins

• Analyze whether peptidoglycan-binding motifs are conserved in EccB2

The periplasmic domain of EccB1 consists of a central domain flanked by repeat domains that form a quasi 2-fold symmetrical structure , which may provide insights into EccB2's structure.

What are the common methods for expressing and purifying recombinant EccB2?

Effective expression and purification of recombinant EccB2 typically follows these methodological approaches:

Expression systems:

• E. coli systems (BL21(DE3), Rosetta) are commonly used for cytoplasmic domains

• Mycobacterium smegmatis expression for better folding of full-length protein

• Baculovirus-infected insect cells for membrane proteins

Expression strategies:

• Use of solubility tags (His, MBP, SUMO) to enhance protein solubility

• Codon optimization for the expression host

• Inducible promoters (T7, tet) for controlled expression

• Lower temperature cultivation (16-20°C) to improve folding

Purification protocol:

• Cell lysis: Sonication or French press

• Initial capture: IMAC for His-tagged proteins

• Intermediate purification: Ion exchange chromatography

• Polishing: Size exclusion chromatography

• Quality control: SDS-PAGE, Western blot, mass spectrometry

For membrane-associated regions:

• Detergent screening (DDM, LDAO, OG) for solubilization

• Amphipol or nanodisc reconstitution for stabilization

According to product information, recombinant EccB2 has been successfully expressed in E. coli with an N-terminal His tag .

What experimental approaches are most effective for studying the function of EccB2 in vitro?

Studying EccB2 function in vitro requires specialized methodological approaches:

Protein-protein interaction studies:

• Pull-down assays using His-tagged EccB2 as bait

• Surface plasmon resonance for measuring binding kinetics

• Isothermal titration calorimetry for thermodynamic parameters

• Crosslinking mass spectrometry to map interaction interfaces

Structural approaches:

Functional reconstitution:

• Liposome reconstitution assays to test membrane integration

• In vitro secretion assays using artificial membrane systems

• Fluorescence-based assays to monitor protein translocation

Biophysical characterization:

• Thermal stability assays to assess structural integrity

• Limited proteolysis to identify flexible regions

• Circular dichroism spectroscopy for secondary structure analysis

These approaches should be combined in a systematic workflow starting with binary protein-protein interactions and progressing to reconstitution of larger subcomplexes, as demonstrated in studies of other ESX systems .

What are the known or predicted interaction partners of EccB2 in the ESX-2 system?

Based on studies of other ESX systems, EccB2 likely interacts with multiple partners within the ESX-2 secretion system:

Core complex components:

• EccC2: The ATPase component that provides energy for secretion

• EccD2: Transmembrane component forming the translocation channel

• EccE2: Membrane-associated component stabilizing the complex

Interaction mapping approaches:

• Co-immunoprecipitation using anti-EccB2 antibodies

• Bacterial two-hybrid for binary protein interactions

• Proximity labeling (BioID or APEX2) fused to EccB2

• Crosslinking-mass spectrometry to capture transient interactions

From structural studies, we know that in ESX-3, "The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein" . This suggests EccB2 likely forms similar stoichiometric relationships with its ESX-2 partners.

When designing experiments with recombinant EccB2, several methodological considerations are critical:

Sample preparation:

• Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Addition of glycerol (final concentration 5-50%) for long-term storage

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

• Aliquoting to avoid freeze-thaw cycles, which significantly impact stability

Experimental controls:

• Include properly folded EccB2 positive control

• Use denatured EccB2 as negative control

• Include other EccB family proteins for specificity testing

Replication strategy:

• Implement biological replicates (different protein preparations)

• Avoid confounding variables by standardizing expression and purification protocols

• Address batch effects through randomized block design

Statistical considerations:

• Power analysis to determine appropriate sample size

• Randomization to minimize bias

• Blinding during data analysis when possible

Specific assay considerations:

• For binding assays: account for non-specific binding

• For structural studies: assess sample homogeneity

• For functional studies: validate activity using multiple approaches

Proper experimental design with sufficient replication is essential, as "biological replicates are absolutely essential" for robust data analysis .

How can I validate the activity of recombinant EccB2 after purification?

Validating EccB2 activity requires multiple complementary approaches:

Structural integrity assessment:

• Circular dichroism spectroscopy to confirm secondary structure content

• Thermal shift assays to measure protein stability

• Size exclusion chromatography to verify oligomeric state

• SDS-PAGE to assess purity (>90% as indicated for commercial preparations)

Functional validation steps:

• Binding assays with other ESX-2 components:

  • Pull-down experiments with purified EccC2, EccD2, EccE2

  • AlphaScreen or ELISA-based interaction assays

  • Microscale thermophoresis to quantify binding affinity

• Complementation assays:

  • Introduction of purified EccB2 into membrane vesicles from EccB2-deficient strains

  • Functional rescue of EccB2 knockout strains

• Assembly assays:

  • Native PAGE to monitor formation of higher-order complexes

  • Electron microscopy to visualize complex assembly

For membrane-associated forms, reconstitution into liposomes or nanodiscs followed by substrate binding assays provides the most physiologically relevant validation.

How can cryo-electron microscopy be optimized for studying the structure of EccB2 within the ESX-2 complex?

Optimizing cryo-EM for EccB2 structural studies involves several critical methodological considerations:

Sample preparation optimization:

• Protein expression and purification:

  • Scale-up production for higher yields

  • Optimize buffer conditions for complex stability

  • Use nanobodies or Fab fragments to stabilize flexible regions

• Complex assembly:

  • Co-expression of multiple components

  • Controlled reconstitution into membrane mimetics

  • Crosslinking strategies to stabilize transient interactions

• Cryo-EM grid preparation:

  • Optimize protein concentration (typically 0.5-5 mg/ml)

  • Screen detergents or amphipols to reduce aggregation

  • Test different grid types (Quantifoil, C-flat)

  • Evaluate additives to improve particle distribution

Data collection parameters:

Image processing approaches:

• Deep learning-based particle picking for heterogeneous samples

• 3D variability analysis to capture conformational states

• Focused refinement targeting specific regions (e.g., EccB2 periplasmic domain)

• Multi-body refinement to account for domain flexibility