Recombinant Aggregatibacter actinomycetemcomitans UPF0070 protein 1057 (1057)
Description
General Information
Recombinant Aggregatibacter actinomycetemcomitans UPF0070 protein 1057 (1057) is a protein derived from the bacterium Aggregatibacter actinomycetemcomitans . A. actinomycetemcomitans is a gram-negative bacterium that is part of the normal oral flora but can also cause invasive infections .
Function and Characteristics of Aggregatibacter actinomycetemcomitans
Aggregatibacter actinomycetemcomitans has several virulence factors that contribute to its pathogenicity . One such factor is the cytolethal distending toxin (Cdt), a heterotrimeric AB2 toxin composed of CdtA, CdtB, and CdtC subunits . The Cdt complex binds to cell surfaces and delivers the active subunit, CdtB, into the cell, leading to cell cycle arrest and apoptosis . Another virulence factor is ApiA, a trimeric autotransporter outer membrane protein (Omp) involved in auto-aggregation, epithelial cell binding, and complement resistance .
Role of Cellugyrin in CdtB Internalization
Research indicates that the internalization of CdtB, the active subunit of the A. actinomycetemcomitans Cdt toxin, depends on cellugyrin, also known as synaptogyrin 2 . Cellugyrin is a tetraspanin membrane protein present in most cells and functions in intracellular transport . Studies have shown that Cdt binding to cholesterol-rich membrane microdomains leads to increased cellugyrin levels and translocation to these microdomains . Cells deficient in cellugyrin are unable to internalize CdtB and are resistant to its toxic effects .
ApiA and Serum Resistance
ApiA, an outer membrane protein of A. actinomycetemcomitans, plays a crucial role in serum resistance by interacting with human Factor H, which disrupts the complement cascade . Specific regions within ApiA are responsible for Factor H binding and complement resistance . Deletion of a region spanning amino acids 186–217 in ApiA abrogates complement resistance and Factor H binding, while a 13-amino-acid peptide promotes serum resistance .
Virulence Variation Among Serotypes
Different serotypes of A. actinomycetemcomitans exhibit variations in virulence . Serotype a isolates show less heterogeneity compared to serotype b or c isolates . Variations in adhesion to epithelial cells and cytotoxic effects have been observed among different serotypes .
Table Summarizing Key Proteins and Their Functions
Product Specs
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Target Background
Q&A
FAQs for Researchers on Recombinant Aggregatibacter actinomycetemcomitans UPF0070 Protein 1057 (1057)
What expression systems are optimal for producing recombinant UPF0070 protein 1057, and how do they impact functional studies?
Recombinant UPF0070 protein 1057 is produced in baculovirus (insect cells) and yeast systems, as demonstrated by product variants CSB-BP524438AYK1 (baculovirus) and CSB-YP524438AYK1 (yeast) . Key considerations:
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Baculovirus systems are preferred for eukaryotic post-translational modifications but require longer production times.
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Yeast systems offer faster production but may lack specific modifications.
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Purity (>85% via SDS-PAGE) and storage conditions (lyophilized vs. liquid) affect protein stability. Lyophilized forms have a 12-month shelf life at -80°C, while liquid forms last 6 months .
How should researchers handle storage and reconstitution to maintain protein integrity?
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Reconstitution: Centrifuge vials before resuspending in sterile water (0.1–1.0 mg/mL). Adding 5–50% glycerol improves long-term stability .
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Storage: Avoid repeated freeze-thaw cycles. Working aliquots stored at 4°C retain stability for ≤1 week .
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Critical step: Verify post-reconstitution activity via SDS-PAGE or functional assays to confirm structural integrity.
What assays are recommended for initial functional characterization of UPF0070 protein 1057?
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Purity validation: SDS-PAGE and Western blotting using species-specific antibodies .
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Functional screening: Partner with virulence studies of A. actinomycetemcomitans outer membrane vesicles (OMVs), which deliver toxins like leukotoxin (LtxA) . UPF0070 may share interaction pathways with OMV-associated proteins involved in immune evasion or nutrient acquisition .
How can structural variations in UPF0070 protein 1057 impact its role in bacterial pathogenesis?
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Comparative analysis: Use circular dichroism (CD) spectroscopy to assess secondary structures (e.g., α-helix content) under different reconstitution conditions, as done for recombinant LtxA .
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Domain mapping: Identify regions critical for interactions with host cells or other bacterial proteins using truncation mutants (see ApiA studies ).
What experimental strategies resolve contradictions in UPF0070’s functional data across studies?
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Case example: If cytotoxicity assays yield inconsistent results, validate whether protein acylation (a post-translational modification) is required for activity, as observed in LtxA .
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Controls: Include non-acylated protein variants and compare bioactivity via neutrophil granule exocytosis assays .
How can UPF0070 protein 1057 be integrated into studies of A. actinomycetemcomitans immune evasion mechanisms?
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Interaction studies: Screen for binding to host complement regulators (e.g., Factor H) using surface plasmon resonance (SPR), a method validated for ApiA .
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OMV proteomics: Co-purify UPF0070 with OMVs using density gradient centrifugation and LC-MS/MS, as described in .
Table 1: Comparison of Recombinant UPF0070 Protein Production Systems
Table 2: Key Virulence-Related Proteins in A. actinomycetemcomitans for Functional Cross-Reference
Methodological Recommendations
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For structural studies: Combine CD spectroscopy with cryo-EM to resolve UPF0070’s tertiary structure .
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For functional assays: Use neutrophil granule exocytosis models to test immunomodulatory effects .
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For data validation: Cross-reference with OMVs proteomics datasets (e.g., ProteomeXchange PXD002509 ).
