Recombinant Pasteurella multocida Uncharacterized protein PM0654 (PM0654)

What is the predicted structure and function of Pasteurella multocida uncharacterized protein PM0654?

PM0654 is an uncharacterized protein from Pasteurella multocida, a Gram-negative bacterium responsible for various diseases in animals, including porcine atrophic rhinitis and fowl cholera. While definitive structural information remains limited, bioinformatic analysis suggests PM0654 contains potential binding domains that may interact with host cellular components. Sequence homology analysis indicates potential structural similarities to bacterial virulence factors, though experimental validation is required. Researchers should employ multiple prediction tools including BLAST, Pfam, and I-TASSER for structural modeling, followed by experimental verification through X-ray crystallography or cryo-electron microscopy. These approaches will help elucidate the three-dimensional structure and provide insights into potential functional domains.

How does PM0654 compare to other characterized proteins in Pasteurella multocida?

Unlike the well-characterized P. multocida toxin (PMT, 146 kDa) that has been established as a key virulence factor in causing lung and turbinate lesions, PM0654 remains largely uncharacterized . PMT has demonstrated roles in pathogenesis through multiple epitopes, with research showing 10 B-cell epitopes and 13 T-cell epitopes identified through bioinformatic analysis . By comparison, PM0654 has not been thoroughly investigated for epitope mapping or functional characterization. Researchers should conduct comparative genomic analysis between PM0654 and other P. multocida proteins to identify conserved domains or motifs that might suggest functional roles. Additionally, gene expression analysis under various growth conditions may reveal co-expression patterns with known virulence factors, potentially indicating functional relationships or involvement in similar pathogenic pathways.

What isolation techniques are most effective for obtaining native PM0654 from Pasteurella multocida cultures?

For isolating native PM0654 from P. multocida cultures, researchers should first establish reliable bacterial culture conditions. P. multocida can be isolated from suspected cases using conventional bacteriological techniques, including cultivation on blood agar, as demonstrated in isolation protocols from clinical samples . For protein isolation specifically, a multi-step purification approach is recommended. Begin with bacterial cell lysis using either sonication or a French press in the presence of protease inhibitors. Differential centrifugation can separate cellular compartments, followed by ammonium sulfate precipitation to concentrate proteins. For higher purity, employ chromatographic techniques such as ion exchange chromatography followed by size exclusion chromatography. Confirmation of protein identity should include Western blotting with antibodies specific to PM0654 or mass spectrometry analysis for definitive identification.

What expression systems are optimal for recombinant production of PM0654?

For recombinant production of PM0654, several expression systems should be evaluated based on research objectives. E. coli remains the first-line expression system due to its rapid growth, high protein yields, and established protocols. BL21(DE3) strains often provide good expression levels for bacterial proteins. For potential toxic proteins, consider using tightly regulated systems like pET vectors with T7 promoters or the arabinose-inducible pBAD system. If post-translational modifications are suspected to be important for PM0654 function, eukaryotic systems such as Pichia pastoris or baculovirus-infected insect cells may be more appropriate. Based on protocols used for other P. multocida recombinant proteins, expression with a GST tag has proven effective, as demonstrated in the successful expression of soluble rPMT protein (97 kDa) containing a GST tag . Expression constructs should be designed with appropriate affinity tags (His6, GST, or MBP) to facilitate purification, with cleavage sites for tag removal if necessary for functional studies.

How should researchers address solubility issues when expressing recombinant PM0654?

Addressing solubility issues for recombinant PM0654 requires a systematic approach. First, conduct expression trials at multiple temperatures (16°C, 25°C, 30°C, 37°C) and IPTG concentrations (0.1-1.0 mM) to identify optimal conditions that minimize inclusion body formation. Evidence from related P. multocida recombinant protein studies indicates that soluble expression can be achieved, as demonstrated with the rPMT protein which was successfully expressed in soluble form . If insolubility persists, consider fusion partners known to enhance solubility such as MBP, SUMO, or Trx. Codon optimization for the expression host may improve translation efficiency and folding. For proteins that remain insoluble, refolding strategies can be employed, including gradual dialysis against decreasing concentrations of denaturants, pulsed dilution, or on-column refolding techniques. Additives such as L-arginine, non-detergent sulfobetaines, or low concentrations of specific detergents may enhance refolding efficiency. Always validate the structural integrity of refolded protein using circular dichroism or limited proteolysis to ensure native-like conformation.

What purification strategy yields the highest purity recombinant PM0654 for structural studies?

A multi-step purification strategy is essential for obtaining high-purity recombinant PM0654 suitable for structural studies. Based on successful approaches with other P. multocida recombinant proteins, initial capture should utilize affinity chromatography matching the fusion tag (e.g., Ni-NTA for His-tagged proteins or glutathione Sepharose for GST-tagged proteins) . Following affinity purification, intermediate purification using ion exchange chromatography separates protein variants with different surface charges. For final polishing, size exclusion chromatography removes aggregates and provides buffer exchange into a stabilizing formulation. For structural biology applications, additional considerations include removal of affinity tags using specific proteases (TEV, thrombin, or PreScission), followed by reverse affinity chromatography. Throughout purification, monitor protein quality using SDS-PAGE, dynamic light scattering to assess monodispersity, and thermal shift assays to identify stabilizing buffer conditions. For crystallography purposes, concentrated protein (typically >10 mg/mL) should undergo pre-crystallization testing to ensure homogeneity and stability prior to crystallization trials.

What are the recommended approaches for determining the potential virulence role of PM0654 in Pasteurella multocida infections?

Determining the potential virulence role of PM0654 requires a multi-faceted approach. Begin with gene knockout studies using homologous recombination or CRISPR-Cas9 to create PM0654-deficient P. multocida strains. Compare the virulence of wild-type and knockout strains in appropriate animal models, similar to methodologies used in P. multocida challenge studies that assessed lung tissue lesions and neutrophil infiltration . For molecular insights, perform protein-protein interaction studies using pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens to identify host or bacterial binding partners. Cellular localization studies using fluorescently tagged PM0654 can reveal subcellular targeting. Additionally, assess the effects of recombinant PM0654 on host cell cultures by measuring cytotoxicity, cellular morphology changes, or altered signaling pathways. Immune response profiling should include quantification of cytokines (similar to IFN-γ and IL-12 measurements in PMT studies) and leukocyte activation to understand host immunological responses . These complementary approaches will provide comprehensive insights into the potential virulence functions of this uncharacterized protein.

How can researchers design epitope mapping experiments for PM0654 to facilitate vaccine development?

Epitope mapping for PM0654 should employ both computational prediction and experimental validation approaches. Begin with in silico analysis using algorithms that predict B-cell epitopes (based on surface accessibility, hydrophilicity, and flexibility) and T-cell epitopes (using MHC binding prediction tools). This bioinformatics approach was successfully employed for PMT, identifying 10 B-cell epitopes, 8 peptides with multiple B-cell epitopes, and 13 T-cell epitopes . For experimental validation, synthesize overlapping peptides spanning the entire PM0654 sequence and screen them using sera from animals infected with P. multocida to identify immunoreactive regions. ELISA, peptide arrays, and surface plasmon resonance can quantify binding affinities. For conformational epitopes, hydrogen-deuterium exchange mass spectrometry or X-ray crystallography of antibody-antigen complexes provide structural insights. T-cell epitope mapping should include MHC-peptide binding assays and T-cell activation studies using peptide-pulsed antigen-presenting cells. Identified epitopes should then be incorporated into a multi-epitope recombinant antigen design, similar to the rPMT approach, which demonstrated excellent immunogenicity in mouse models . Immunization trials should measure antibody titers, splenocyte proliferation, cytokine profiles, and most importantly, protection against challenge with virulent P. multocida strains.

What cellular and biochemical assays are most informative for characterizing the enzymatic activity of PM0654?

To characterize potential enzymatic activities of PM0654, a systematic screening approach is recommended. Begin with sequence-based predictions to identify potential catalytic motifs or domains that suggest specific enzymatic functions. Based on these predictions, design focused biochemical assays targeting likely activities such as proteolysis, glycosidase activity, lipase activity, or nucleic acid modification. For each potential activity, employ substrate screening using chromogenic or fluorogenic substrates, monitoring spectrophotometric changes upon substrate conversion. Enzymatic parameters (Km, Vmax, kcat) should be determined under varying conditions (pH, temperature, ion concentrations) to establish optimal reaction parameters. Structure-function relationships can be explored through site-directed mutagenesis of predicted catalytic residues, comparing wild-type and mutant protein activities. For cellular activity assessment, introduce purified PM0654 to relevant cell types and monitor phenotypic changes, subcellular alterations, or biochemical modifications. Mass spectrometry-based approaches can identify post-translational modifications induced by PM0654 or detect metabolic changes in treated cells. These combined approaches will provide comprehensive characterization of potential enzymatic functions.

How should researchers design gene knockout experiments to study PM0654 function within the context of Pasteurella multocida pathogenesis?

For gene knockout studies of PM0654, researchers should employ either homologous recombination or CRISPR-Cas9 systems adapted for use in P. multocida. When designing homologous recombination constructs, include at least 1 kb homology arms flanking a selectable marker (typically antibiotic resistance). For CRISPR-Cas9, design guide RNAs with minimal off-target effects and provide a repair template for precise editing. Consider creating both complete knockouts and conditional systems using inducible promoters to study essential genes. Prior to conducting virulence studies, thoroughly characterize knockout strains through growth curve analysis, morphological examination, and comparative proteomics to identify compensatory mechanisms. Virulence studies should employ appropriate animal models reflecting natural host species, such as mice or chickens for P. multocida . Evaluate multiple virulence parameters including colonization efficiency, tissue distribution, persistence, and host immune response profiles. Additionally, perform comparative transcriptomics between wild-type and knockout strains under infection-relevant conditions to identify dysregulated pathways. Competition assays co-infecting with wild-type and knockout strains can reveal subtle fitness defects. This comprehensive approach will provide insights into PM0654's role within the broader context of P. multocida pathogenesis mechanisms.

What structural biology techniques would best elucidate the three-dimensional structure of PM0654, and what sample preparation considerations are critical?

Elucidating PM0654's structure requires selection of appropriate structural biology techniques based on protein properties. X-ray crystallography remains the gold standard for atomic-level resolution but requires well-diffracting crystals. For crystallization, prepare highly pure (>95%) monodisperse protein at 5-15 mg/mL and screen hundreds of conditions varying precipitants, buffers, and additives. Cryo-electron microscopy (cryo-EM) offers an alternative approach particularly valuable for flexible proteins or membrane-associated complexes, requiring optimization of grid preparation and vitrification conditions. For smaller domains (<25 kDa), nuclear magnetic resonance (NMR) spectroscopy can provide structure and dynamics information in solution, necessitating isotopic labeling (15N, 13C) of recombinant protein. Critical sample preparation considerations include buffer optimization through thermal shift assays to identify stabilizing conditions, removal of flexible regions that might impede crystallization through limited proteolysis and construct optimization, assessment of glycosylation or other post-translational modifications that affect homogeneity, and evaluation of protein oligomeric state through size exclusion chromatography coupled with multi-angle light scattering. Surface entropy reduction through mutation of surface-exposed flexible residues (typically clusters of Lys/Glu) can enhance crystallization propensity. Throughout structural studies, iterative refinement of constructs based on preliminary structural data will maximize chances of success.

How can researchers develop a multi-epitope vaccine incorporating PM0654 epitopes, and what immunological parameters should be monitored?

Development of a multi-epitope vaccine incorporating PM0654 epitopes should follow a systematic approach similar to the successful rPMT vaccine strategy . Begin with comprehensive epitope prediction and experimental validation as described previously, focusing on both B-cell and T-cell epitopes. For vaccine construction, selected epitopes should be joined using flexible linkers (GPGPG for B-cell epitopes, AAY for T-cell epitopes) to preserve independent folding and accessibility. Consider incorporating promiscuous T-helper epitopes to enhance immune responses across diverse MHC backgrounds. Express the recombinant multi-epitope construct in an appropriate system (E. coli yielded soluble protein for rPMT ) and purify to homogeneity. For immunization studies, formulate with appropriate adjuvants (alum, oil-in-water emulsions, or TLR agonists) and establish dose-response relationships. Immunological parameters to monitor should include:

• Humoral immunity: Serum IgG titers, antibody subclass distribution, antibody avidity maturation, neutralization capacity

• Cell-mediated immunity: Splenocyte proliferation in response to antigen stimulation, cytokine profiles (particularly IFN-γ and IL-12, which were significantly upregulated in rPMT studies )

• Memory response: Long-term antibody persistence, memory B and T cell quantification

• Protection efficacy: Challenge studies with virulent P. multocida strains, measuring survival rates (the rPMT vaccination showed 57.1% survival rate ), bacterial clearance, and histopathological assessment of target tissues

The vaccine formulation should be iteratively optimized based on these immunological readouts, with particular attention to cellular immune responses that correlate with protection.

What NIH guidelines must researchers follow when working with recombinant Pasteurella multocida proteins like PM0654?

Researchers working with recombinant P. multocida proteins must comply with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules . These guidelines define recombinant nucleic acids as "molecules that are constructed by joining nucleic acid molecules and that can replicate in a living cell" . Key compliance requirements include:

• Institutional Biosafety Committee (IBC) approval before initiation of research

• Implementation of appropriate biosafety practices and containment principles

• Risk assessment based on the characteristics of the organism and the recombinant construct

• Adherence to laboratory containment levels (typically BSL-2 for P. multocida)

• Proper documentation and reporting procedures

The guidelines apply to all recombinant or synthetic nucleic acid research conducted at or sponsored by institutions receiving NIH support, as well as testing in humans of materials containing recombinant or synthetic nucleic acids developed with NIH funds . For international collaborations, researchers must comply with host country rules if established, or have the research reviewed and approved by an NIH-approved IBC and accepted by appropriate national governmental authorities if no local rules exist . Researchers should regularly consult the most current version of the NIH Guidelines, as they are periodically updated to reflect advances in scientific knowledge and risk assessment methodologies.

What are the critical quality control parameters for ensuring reproducibility in PM0654 recombinant protein production for research applications?

Ensuring reproducibility in PM0654 recombinant protein production requires rigorous quality control at multiple stages. During expression, monitor induction efficiency through SDS-PAGE and Western blot analysis, comparing pre- and post-induction samples. For purification, maintain detailed records of all chromatography conditions, including column types, buffer compositions, flow rates, and elution gradients. Critical quality control parameters to assess in the final protein preparation include:

• Purity assessment: >95% purity by SDS-PAGE and size exclusion chromatography

• Identity confirmation: Mass spectrometry analysis for molecular weight verification and peptide mapping

• Structural integrity: Circular dichroism for secondary structure content, fluorescence spectroscopy for tertiary structure assessment

• Homogeneity: Dynamic light scattering to confirm monodispersity and absence of aggregation

• Endotoxin levels: Limulus Amebocyte Lysate (LAL) assay, particularly important for immunological studies

• Functional activity: Develop specific activity assays based on predicted or established functions

• Stability assessment: Accelerated stability studies and freeze-thaw cycle testing

For batch-to-batch consistency, establish acceptable ranges for each parameter and maintain detailed production records. Implement a reference standard system where early, well-characterized batches serve as comparators for subsequent productions. Proper storage conditions (-80°C for long-term, with minimal freeze-thaw cycles) and standardized aliquoting procedures are essential for maintaining protein integrity across experiments.