Recombinant Ureaplasma parvum serovar 3 Uncharacterized protein UU044 (UU044)

What is the taxonomic classification of Ureaplasma parvum serovar 3?

Ureaplasma parvum (previously classified as Ureaplasma urealyticum biovar 1) is one of two species that resulted from the division of what was formerly known as Ureaplasma urealyticum. Taxonomic studies have established that U. parvum can be separated into three subtypes, represented by serovars 1, 3/14, and 6. Epidemiological studies have shown that among U. parvum strains, serovars 3/14 (48%) and 1 (43%) are found more commonly than serovar 6 (23%) in clinical isolates. This classification is based on multiple-banded antigen (MBA) gene variation, which has been key to developing PCR-based typing systems for identification and subtyping .

How does UU044 differ structurally from other proteins in U. parvum serovar 3?

UU044 is a full-length protein (782 amino acids) with a specific sequence profile that distinguishes it from other proteins in U. parvum serovar 3. The protein contains a complex amino acid sequence with multiple domains and potential functional motifs. Analysis of its primary structure shows a sequence rich in lysine residues and repetitive elements (particularly KP motifs in the N-terminal region), which suggests potential roles in protein-protein interactions or structural functions. The complete amino acid sequence begins with "MKFIKRKTKLLTITIGAVAVSSILLGGIFYGTSQKSPSSFGIASIDQKENFINKDNLDYQ..." and continues through 782 residues as documented in recombinant protein specifications .

What methods are available for detecting and identifying U. parvum serovar 3 in clinical samples?

Detection and identification of U. parvum serovar 3 in clinical samples can be performed using PCR-based approaches targeting specific genetic regions. Methods include:

• Species-specific PCR: Primer pairs such as UPS1-UPA, UPS1-UPA1, UPS-UPSA, UPS2-UPA2, and UMS-57–UMA222 are specific for U. parvum and can amplify all 4 serovars of this species.

• Serovar-specific PCR: For specific identification of serovar 3, primer pairs such as UMS-125–UMA269 can be used, which selectively amplify serovar 3 or 14 DNA.

• Direct specimen analysis: PCR-based typing can be applied directly to clinical specimens, eliminating the need for culture isolation. In studies examining vaginal swabs, this approach successfully identified and subtyped ureaplasmas with high sensitivity.

These molecular methods offer significant advantages over conventional serotyping in terms of speed, specificity, and the ability to process clinical specimens directly .

What expression systems are optimal for producing recombinant UU044 protein?

The optimal expression system for recombinant UU044 depends on research objectives and downstream applications. Available data indicates several viable options:

Current commercial recombinant preparations of UU044 utilize E. coli expression systems with N-terminal His-tags, which has proven successful for producing the full-length 782 amino acid protein . For most research applications, this approach provides sufficient quantity and quality of protein, particularly for initial characterization studies.

What purification strategies yield the highest purity of recombinant UU044?

Achieving high purity recombinant UU044 typically requires a multi-step purification strategy:

• Initial capture: For His-tagged UU044, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides effective initial purification.

• Intermediate purification: Ion-exchange chromatography can remove contaminants with different charge properties from the target protein.

• Polishing step: Size exclusion chromatography separates the target protein from aggregates and smaller contaminants.

• Buffer optimization: The final product should be buffer-exchanged into a stabilizing formulation, often containing trehalose (6%) in Tris/PBS-based buffer at pH 8.0 .

This approach can achieve >90% purity as determined by SDS-PAGE, sufficient for most research applications. For specialized applications requiring higher purity, additional chromatographic steps or selective precipitation methods may be employed.

How should UU044 protein be stored to maintain stability and functionality?

Proper storage of recombinant UU044 is essential for maintaining its stability and functionality over time. Based on available product information:

• Long-term storage: Store lyophilized protein at -20°C/-80°C upon receipt. For reconstituted protein, add glycerol (5-50% final concentration) and store in aliquots at -20°C/-80°C.

• Working stocks: Store aliquots at 4°C for up to one week to avoid repeated freeze-thaw cycles.

• Reconstitution protocol: Briefly centrifuge the vial prior to opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Stability considerations: Repeated freezing and thawing should be avoided as it can lead to protein denaturation, aggregation, and loss of activity .

These storage recommendations ensure maintained protein integrity for experimental applications while minimizing degradation or loss of potential functional activity.

What approaches can determine the subcellular localization of UU044 in U. parvum cells?

Determining the subcellular localization of UU044 requires specialized techniques due to the small size of Ureaplasma cells. Effective methodological approaches include:

• Immunoelectron microscopy: Using antibodies raised against recombinant UU044 with gold particle conjugation to visualize the protein's location at ultrastructural level.

• Fractionation studies: Separating membrane, cytoplasmic, and potential periplasmic fractions of Ureaplasma cells followed by Western blotting to detect UU044.

• Fluorescent protein fusion: Generating recombinant Ureaplasma expressing UU044 fused to fluorescent reporters, though genetic manipulation of Ureaplasma remains challenging.

• Protease shaving: Treating intact cells with proteases that cannot penetrate the membrane, then analyzing which proteins/domains are digested to determine surface exposure.

Analysis of the UU044 amino acid sequence reveals potential transmembrane domains and signal sequences that suggest possible membrane association, though experimental validation is necessary to confirm these predictions.

How can researchers investigate potential interactions between UU044 and host proteins?

Investigating UU044-host protein interactions requires a systematic approach combining multiple complementary methods:

• Affinity-based identification:

  • Pull-down assays using His-tagged UU044 as bait against host cell lysates

  • Co-immunoprecipitation from infected host cells

  • Protein microarrays to screen for interactions with specific host protein families

• Validation of identified interactions:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Microscale thermophoresis (MST) for quantitative interaction analysis

  • ELISA-based binding assays for high-throughput confirmation

• Functional significance assessment:

  • Competitive inhibition assays using antibodies or peptides

  • Mutagenesis of predicted interaction interfaces

  • siRNA knockdown of candidate host proteins to assess functional outcomes

This multi-method approach provides both identification and validation of potential interaction partners while offering insights into their functional significance in host-pathogen biology.

What are the most effective methods for generating antibodies against UU044?

Generating specific antibodies against UU044 requires careful consideration of antigen preparation and immunization strategies:

• Antigen selection:

  • Full-length recombinant protein (optimal for polyclonal antibodies)

  • Synthetic peptides corresponding to predicted epitopes (15-25 amino acids)

  • Unique domains with high predicted antigenicity

• Host selection and immunization protocol:

  • Rabbits: For polyclonal antibodies against full-length protein

  • Mice: For monoclonal antibody development

  • Initial immunization with complete Freund's adjuvant followed by boosters with incomplete adjuvant

• Antibody purification:

  • Protein A/G affinity chromatography for IgG isolation

  • Antigen-specific affinity purification using immobilized UU044

• Validation methods:

  • Western blotting against recombinant protein and native Ureaplasma lysates

  • Immunofluorescence microscopy using fixed Ureaplasma cells

  • ELISA titering against pure antigen

  • Epitope mapping to confirm specificity

Recombinant UU044 produced in E. coli with His-tag purification provides suitable antigen material for immunization, though considerations for protein folding may impact epitope recognition in native contexts.

How can researchers investigate the potential role of UU044 in U. parvum pathogenesis?

Investigating UU044's potential role in pathogenesis requires a systematic research approach:

• Expression analysis during infection:

  • RT-qPCR to measure UU044 transcript levels during different phases of infection

  • Proteomic analysis of Ureaplasma recovered from experimental infections

  • Immunohistochemistry on infected tissues to detect UU044 expression in vivo

• Comparative genomics and expression:

  • Analysis of UU044 sequence conservation across clinical isolates associated with different disease severities

  • Comparison of expression levels between strains with different virulence profiles

  • Identification of potential regulatory elements affecting UU044 expression

• Functional studies:

  • Development of UU044 knockout or knockdown systems (challenging in Ureaplasma)

  • Complementation studies to verify phenotype restoration

  • Heterologous expression in model bacteria to assess specific functions

• Host response analysis:

  • Measurement of inflammatory markers in response to purified UU044

  • Evaluation of host cell transcriptional responses to UU044 exposure

  • Assessment of UU044 immunogenicity in clinical samples

This integrated approach can reveal whether UU044 contributes to the pathogenicity of U. parvum serovar 3, which is frequently isolated in clinical settings (48% of U. parvum isolates) .

What structural biology approaches are most suitable for UU044 characterization?

Characterizing the three-dimensional structure of UU044 requires selection of appropriate methods based on protein properties:

The most effective strategy likely involves a combination of these approaches, starting with domain identification and construct optimization based on the full amino acid sequence .

How can differential expression of UU044 under varying conditions be accurately measured?

Accurately measuring UU044 expression under different experimental conditions requires careful selection of methodologies:

• Transcriptional analysis:

  • RT-qPCR with carefully validated reference genes specific for Ureaplasma

  • RNA-Seq for genome-wide expression context

  • Normalization using multiple reference genes to account for experimental variation

• Protein-level quantification:

  • Western blotting with densitometry (semi-quantitative)

  • ELISA development using anti-UU044 antibodies

  • Selected Reaction Monitoring (SRM) mass spectrometry for absolute quantification

• Statistical considerations:

  • Minimum of 3-5 biological replicates per condition

  • Appropriate statistical tests based on data distribution (t-test, ANOVA, non-parametric alternatives)

  • Multiple testing correction for genome-wide analyses

• Experimental design optimization:

  • Time-course sampling to capture expression dynamics

  • Controlled growth conditions to minimize variables

  • Inclusion of appropriate positive and negative controls

These approaches allow reliable quantification of UU044 expression changes in response to environmental factors, growth phases, or host cell interactions.

How should researchers analyze sequence variations in UU044 across clinical isolates?

Analysis of UU044 sequence variations across clinical isolates requires rigorous methodological approaches:

• Sequence alignment and conservation analysis:

  • Multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Conservation scoring to identify highly conserved residues/motifs

  • Visualization using tools like Jalview or WebLogo

• Polymorphism characterization:

  • Calculation of nucleotide diversity (π) and polymorphism statistics

  • Identification of synonymous vs. non-synonymous substitutions

  • Detection of insertion/deletion events

• Correlation with clinical outcomes:

  • Statistical association between specific variants and disease presentations

  • Multivariate analysis to account for confounding factors

  • Longitudinal analysis for persistent infections

• Evolutionary analysis:

  • Phylogenetic tree construction to visualize relationships between variants

  • Selection pressure analysis (dN/dS ratios) to identify regions under selection

  • Recombination detection to identify potential horizontal transfer events

These approaches can reveal whether specific UU044 variants are associated with particular disease manifestations or enhanced virulence, providing insights into structure-function relationships.

What bioinformatic approaches can predict potential functions of UU044?

Predicting potential functions of uncharacterized proteins like UU044 requires sophisticated bioinformatic analyses:

• Sequence-based approaches:

  • Homology detection using PSI-BLAST and HHpred against diverse databases

  • Motif scanning using PROSITE, PFAM, and other motif databases

  • Disorder prediction to identify structured domains vs. flexible regions

• Structure-based predictions:

  • Ab initio or template-based 3D structure prediction (AlphaFold2, RoseTTAFold)

  • Structural alignment against known protein structures

  • Active site prediction based on structural features

• Network-based approaches:

  • Genomic context analysis (gene neighborhood, operons)

  • Co-expression network analysis if transcriptomic data is available

  • Phylogenetic profiling to identify co-evolving proteins

• Integration and validation:

  • Consensus functional prediction from multiple tools

  • Confidence scoring based on agreement between methods

  • Experimental design to test specific functional hypotheses

This comprehensive bioinformatic strategy can generate testable hypotheses about UU044 function, directing experimental efforts more efficiently than purely empirical approaches.

How can researchers distinguish between direct and indirect effects when studying UU044's impact on host cells?

Distinguishing direct from indirect effects of UU044 on host cells presents a methodological challenge requiring controlled experimental approaches:

• Purified protein studies:

  • Exposure of host cells to purified recombinant UU044 at physiologically relevant concentrations

  • Use of heat-inactivated or protease-treated UU044 as controls

  • Inclusion of polymyxin B to neutralize potential LPS contamination

• Time-course analyses:

  • Monitoring cellular responses at multiple timepoints (minutes to hours)

  • Early responses more likely reflect direct effects

  • Later responses may represent secondary signaling cascades

• Molecular interaction validation:

  • Direct binding assays between UU044 and candidate host receptors

  • Competitive inhibition using antibodies or peptides

  • Receptor knockdown or knockout to verify specificity

• Signaling pathway dissection:

  • Specific inhibitors of key signaling nodes

  • Phosphorylation state analysis of signaling intermediates

  • Reporter constructs for specific transcription factors

These methodological approaches help establish causality and mechanism rather than mere association, critical for understanding UU044's true biological functions in host-pathogen interactions.