Recombinant Mannheimia succiniciproducens UPF0234 protein MS1759 (MS1759)
Description
Introduction to MS1759
MS1759 is a recombinant protein encoded by the MS1759 gene in Mannheimia succiniciproducens MBEL55E, a capnophilic Gram-negative bacterium renowned for its high-efficiency succinic acid production . It belongs to the UPF0234 family of uncharacterized proteins, a group of conserved bacterial proteins with unknown functions . UPF0234 proteins are typically small (e.g., ~200–300 amino acids), lack defined catalytic motifs, and are hypothesized to play roles in stress response, metabolic regulation, or protein-protein interactions .
Biochemical Characteristics
While direct experimental data on MS1759 is scarce in publicly available literature, inferences can be drawn from its classification and the organism’s metabolic context:
| Feature | UPF0234 Family | MS1759 (Inferred) |
|---|---|---|
| Length | ~150–250 aa | ~200–300 aa (estimated) |
| Conservation | High across bacteria | Present in M. succiniciproducens |
| Function | Unknown; potential regulatory or structural | Uncharacterized |
| Localization | Cytoplasmic/membrane-associated | Cytoplasmic (likely) |
Key Observations:
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Genomic Context: UPF0234 proteins often cluster with genes involved in stress response or metabolic adaptation . In M. succiniciproducens, MS1759 may interact with pathways critical for CO₂ utilization or succinic acid synthesis .
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Structural Predictions: UPF0234 proteins typically lack catalytic domains, suggesting non-enzymatic roles (e.g., scaffolding, transcriptional regulation) .
A. Metabolic Context in M. succiniciproducens
M. succiniciproducens MBEL55E produces succinic acid via a reductive TCA cycle, driven by enzymes like PEP carboxykinase, malate dehydrogenase (MDH), and fumarate reductase . While MS1759 is not directly implicated in these pathways, its potential role in:
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Stress Adaptation: UPF0234 proteins may modulate cellular responses to high CO₂ or low pH, common in industrial fermentations .
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Metabolic Regulation: Interaction with transcription factors or chaperones to optimize succinic acid yield .
Comparative Analysis with Other Proteins
| Protein | Function | MS1759 Relevance |
|---|---|---|
| Sucrose PTS (MS0784) | Sucrose transport and phosphorylation | Unrelated to carbohydrate metabolism |
| MDH (Malate Dehydrogenase) | Oxaloacetate reduction for succinic acid | Potential regulatory co-factor |
| Mannose PTS (MS0616-MS0618) | Fructose transport | No overlap in function or sequence |
Notable Absence: MS1759 lacks homology to enzymes in the reductive TCA cycle or sugar metabolism, suggesting a non-catalytic role .
Potential Applications
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Strain Engineering: Overexpression or knockout studies to assess effects on succinic acid yield under stress conditions .
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Protein Interactome Mapping: Identification of binding partners to elucidate regulatory networks .
Technical Challenges
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Low Conservation: Limited homology to functionally characterized proteins complicates functional prediction .
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Experimental Data Deficit: No published studies directly addressing MS1759’s role in M. succiniciproducens .
Future Directions
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Structural Studies: X-ray crystallography or cryo-EM to resolve MS1759’s tertiary structure and identify binding motifs .
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Omics Integration: Metabolomic and proteomic profiling under CO₂-rich conditions to link MS1759 expression to metabolic fluxes .
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Heterologous Expression: Production in E. coli or C. glutamicum to test cross-species functionality .
Product Specs
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Q&A
What is the basic structure and classification of the MS1759 protein?
MS1759 is a 163 amino acid protein from Mannheimia succiniciproducens strain MBEL55E, classified as a member of the UPF0234 family. The protein's sequence was determined through the genome sequencing of M. succiniciproducens . UPF0234 stands for Uncharacterized Protein Family 0234, indicating that while the protein has been identified and sequenced, its specific biological function remains to be fully elucidated. Structural analysis through databases like AlphaFoldDB suggests that MS1759 contains two domains with characteristic YajQ-like superfamily folds .
What is known about the genomic context of MS1759 in Mannheimia succiniciproducens?
MS1759 is encoded by the MS1759 gene in the M. succiniciproducens MBEL55E genome. The gene is part of the complete genome sequence determined by Hong et al. in 2004 . The genomic context analysis of M. succiniciproducens revealed that it has a relatively small genome (2,314,078 bp) with a high proportion of genes involved in carbohydrate transport and metabolism, which supports its efficient succinic acid production capabilities . Although specific operon information for MS1759 is limited in the provided data, genomic context analysis is crucial for understanding potential functional relationships with neighboring genes.
What expression systems are most effective for recombinant production of MS1759?
Based on research with M. succiniciproducens proteins and similar bacterial proteins, E. coli-based expression systems are commonly employed for recombinant production. For MS1759 specifically, both T7-based expression systems and autoinduction methods could be effective . When considering temperature sensitivity of protein folding, the cspA promoter system may be advantageous compared to the tac promoter system, especially at lower temperatures (25°C), which showed improved solubility for aggregation-prone proteins . For optimal expression, a defined autoinduction medium containing a mixture of glucose, glycerol, and lactose as carbon substrates with ammonium as the sole nitrogen source has been developed for similar recombinant proteins, achieving yields of approximately 500 mg L⁻¹ .
What are the methodological considerations for isotope labeling of MS1759 for structural studies?
For researchers interested in structural studies of MS1759 using NMR or other techniques requiring isotope labeling, a protocol based on defined media can achieve high incorporation rates of isotopes. Specifically:
| Isotope Label | Incorporation Rate |
|---|---|
| ¹⁵N | 99% |
| ¹³C | 97% |
| ²H | ~75% |
| Se-Met | 70-90% (depending on strain) |
To achieve these incorporation rates, cultivation should be performed in defined media containing the appropriate isotope-labeled precursors. For selenomethionine labeling, using a methionine auxotrophic E. coli strain can increase incorporation to approximately 90% compared to 70% in prototrophic strains . The protocol involves a simple transition from growth to production phase, with specific media composition adjustments to accommodate the labeled compounds.
What approaches can be used to determine the function of the largely uncharacterized MS1759 protein?
Given the limited functional information available for UPF0234 family proteins, several complementary approaches are recommended:
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Comparative genomics: Analyze similar proteins in related organisms with known functions. Use tools like STRING database (shown in MS1759 entry ) to identify potential protein-protein interactions.
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Structural analysis: Utilize the available structural predictions from AlphaFoldDB and Gene3D databases to identify potential functional sites and domains.
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Transcriptomic correlation: Analyze under which conditions MS1759 is co-expressed with proteins of known function in M. succiniciproducens, particularly during different growth phases as shown in proteome analysis studies .
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Knockout studies: Generate MS1759 knockout strains following methodologies used for other M. succiniciproducens genes as described by Lee et al. , then analyze phenotypic changes, especially related to succinic acid metabolism.
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Protein interaction studies: Use surface plasmon resonance methods to detect potential binding partners, applying experimental designs that can differentiate between specific and non-specific interactions .
How might MS1759 relate to the metabolic capabilities of M. succiniciproducens?
While direct evidence linking MS1759 to specific metabolic pathways in M. succiniciproducens is not explicitly mentioned in the search results, the broader context suggests possible areas for investigation. M. succiniciproducens is primarily studied for its efficient succinic acid production through its anaerobic fermentative metabolism .
Key metabolic pathways identified in M. succiniciproducens include:
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PEP carboxylation (the major CO₂-fixing step)
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Branched tricarboxylic acid cycle
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Sucrose and other carbohydrate utilization pathways, including various PTS (phosphotransferase system) transporters
Researchers should investigate whether MS1759 plays a role in any of these pathways, potentially through proteomic analyses under various carbon source conditions, similar to the approaches used in previous M. succiniciproducens studies .
How can protein-protein interaction kinetics be reliably measured for MS1759?
For analyzing MS1759 interaction kinetics, surface plasmon resonance (SPR) detection provides a powerful approach. Based on experimental design principles for protein-protein interactions:
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Surface preparation: Immobilize MS1759 to surfaces with and without dextran matrix, ensuring use of a reference surface to separate binding signals from matrix conformation changes.
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Data quality improvement: Separate signals related to binding events from those due to differences in refractive index between sample and running buffer.
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Binding curve analysis: Collect data sets with different potential binding partner concentrations and analyze using numerical integration of differential rate equations and global fitting.
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Reaction scheme validation: Since data analysis alone is insufficient to discriminate between different reaction schemes (parallel, competitive, two-state reactions), design critical experiments including:
This comprehensive approach will help overcome common pitfalls in protein interaction studies and provide reliable kinetic parameters.
What advanced proteomic approaches are most suitable for studying MS1759 in the context of the M. succiniciproducens proteome?
For comprehensive proteomic analysis involving MS1759, researchers should consider Top-Down Data-Independent Acquisition Mass Spectrometry (TD-DIA-MS) approaches, which are particularly effective for proteoform identification:
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Software utilization: Implement TopDIA software, which generates demultiplexed pseudo MS/MS spectra from TD-DIA-MS data, enabling comprehensive proteoform identification .
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Database search strategy: Search against the M. succiniciproducens proteome sequence database using tools like TopPIC, allowing for variable post-translational modifications (oxidation, methylation, acetylation, phosphorylation) and unknown mass shifts .
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Mass accuracy parameters: Set error tolerances for precursor and fragment masses to 10 ppm for optimal identification .
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Comparative approach: Implement both TD-DIA-MS and TD-DDA-MS (Data-Dependent Acquisition) approaches for comprehensive coverage, as they show complementary strengths in proteoform identification .
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Membrane protein analysis: For MS1759, specifically analyze whole cellular proteins, membrane proteins, and secreted proteins separately, as was done in previous M. succiniciproducens proteome studies .
How might MS1759 be relevant to metabolic engineering of M. succiniciproducens for improved succinic acid production?
To investigate MS1759's potential role in metabolic engineering applications:
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Genome-scale metabolic model integration: Incorporate MS1759 into the existing genome-scale metabolic network of M. succiniciproducens, which consists of 686 reactions and 519 metabolites , to predict its potential impact on metabolic flux.
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Constraints-based flux analysis: Perform in silico knockout studies of MS1759 to predict its effect on succinic acid production pathways, similar to previous studies that successfully identified gene targets for enhanced succinic acid production .
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Key metabolic characteristics to investigate in relation to MS1759:
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Knockout validation experiments: If in silico predictions suggest MS1759 affects succinic acid production, generate knockout strains following methodologies used for other M. succiniciproducens genes, such as those in the sucrose utilization pathway .
What techniques are available for studying MS1759 protein labeling in complex cellular contexts?
Recent advances in protein labeling technologies offer powerful approaches for studying MS1759 in its native cellular context:
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CuRVE technology: This MIT-developed technology enables labeling of proteins across millions of individual cells in fully intact 3D tissues with unprecedented speed, uniformity, and versatility. The technique allows for labeling whole tissue samples in a single day, revealing insights at the single-cell level .
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Key technical components:
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Application to MS1759 studies: This approach could be particularly valuable for studying MS1759 expression patterns across different cell types within bovine rumen samples, potentially revealing contextual information about its function that would be lost in traditional proteomic analyses .
How does MS1759 compare to homologous proteins in other bacterial species?
MS1759 belongs to the UPF0234 family, with homology to YajQ-like proteins found across bacterial species. Comparative analysis should focus on:
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Sequence conservation: MS1759 has structural similarity to proteins in the YajQ-like superfamily, containing characteristic domain structures identified through Gene3D (3.30.70.860 and 3.30.70.990) and other databases .
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Functional insights from homologs: While MS1759's function remains uncharacterized, related proteins in other bacteria may provide clues. For example, in E. coli, YajQ-like proteins have been implicated in RNA binding and potential regulatory functions.
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Evolutionary context: Analysis through databases like HOGENOM (CLU_099839_1_0_6) and OrthoDB (1612988at2) can provide evolutionary insights into conservation patterns across bacterial lineages .
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Comparative genomic neighborhood: Analysis of genes adjacent to MS1759 homologs in other bacterial species may reveal conserved operons or functional units that could suggest potential roles.
What can be learned from comparing the MS1759 protein to homologous proteins in the prion protein gene family?
While MS1759 and prion proteins belong to different protein families with distinct functions, comparative protein analysis methodologies can be informative:
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Polymorphism analysis: Similar to how the Met/Val polymorphism at codon 129 in prion proteins affects function and disease susceptibility , identifying key polymorphic sites in MS1759 across different M. succiniciproducens strains could provide functional insights.
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Structural comparison approaches: Techniques used to analyze structural variants in prion proteins can be applied to study potential structural variations in MS1759.
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Geographic distribution analysis: Methods used to analyze geographical distribution of prion protein variants could be applied to study the distribution of MS1759 variants in M. succiniciproducens isolated from different geographical regions.
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Disease association methodologies: While MS1759 is not known to be associated with diseases, methodologies used to study disease associations in prion proteins could be adapted to investigate potential phenotypic effects of MS1759 variations.
