Recombinant Uncharacterized protein SMU_988 (SMU_988)

What is SMU_988 and what organism does it come from?

SMU_988 is an uncharacterized protein from Streptococcus mutans serotype c, assigned the UniProt ID P34001 . S. mutans is a gram-positive, facultatively anaerobic bacterium well-known for its role in dental caries (tooth decay). The protein consists of 461 amino acids and has been produced recombinantly with an N-terminal His-tag to facilitate purification and downstream applications .

What structural features or domains characterize SMU_988?

While the complete structural characterization of SMU_988 remains limited in the current literature, the protein sequence analysis suggests it contains transmembrane regions (indicated by hydrophobic amino acid stretches such as "LLVILPFPILGTLFLIYT") . The protein is uncharacterized, meaning its specific functional domains have not been fully annotated or characterized experimentally.

What expression systems are recommended for recombinant SMU_988 production?

Based on available literature, recombinant SMU_988 has been successfully expressed in E. coli expression systems . This heterologous expression system provides several advantages:

• High protein yield

• Well-established protocols

• Compatibility with N-terminal His-tag fusion

• Cost-effectiveness for research applications

The full-length protein (amino acids 1-461) is typically expressed with an N-terminal His-tag to facilitate downstream purification processes .

What purification protocol yields the highest purity for recombinant SMU_988?

The recommended purification protocol for His-tagged SMU_988 follows standard immobilized metal affinity chromatography (IMAC) methodology:

• Lyse E. coli cells expressing the recombinant protein

• Clarify lysate by centrifugation

• Load supernatant onto Ni-NTA or similar metal affinity resin

• Wash with increasing imidazole concentrations to remove non-specific binding

• Elute purified protein with high imidazole buffer

• Perform buffer exchange to Tris/PBS-based buffer with 6% Trehalose, pH 8.0

This protocol typically yields >90% purity as determined by SDS-PAGE analysis .

What is the optimal reconstitution procedure for lyophilized SMU_988?

The recommended reconstitution procedure is as follows:

• Centrifuge the vial briefly before opening to collect all material at the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended final concentration is 50%)

• Aliquot to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for long-term storage

This procedure helps maintain protein stability and activity for experimental applications.

What is the known or predicted function of SMU_988 in S. mutans biology?

While the specific function of SMU_988 remains largely uncharacterized , contextual research suggests it may be involved in bacterial gene regulation networks in Streptococcus mutans . The protein may participate in one or more of the following processes based on research in related S. mutans proteins:

• Metal ion homeostasis (particularly manganese regulation)

• Stress response mechanisms

• Virulence factor expression

• Cell envelope biogenesis

Further functional characterization studies are needed to elucidate its precise role.

What experimental approaches are recommended for functional characterization of SMU_988?

Based on methodologies employed for similar uncharacterized bacterial proteins, the following experimental approaches are recommended:

• Gene knockout or CRISPR-based gene editing to assess phenotypic changes

• Protein-protein interaction studies using pull-down assays

• Subcellular localization using fluorescently-tagged variants

• Gene expression analysis under various environmental conditions

• Structural studies using X-ray crystallography or cryo-EM

• Comparative genomics and phylogenetic analysis

These approaches would help establish the biological context and function of SMU_988.

How does SMU_988 potentially relate to small RNA research in S. mutans?

Recent research on S. mutans has identified small regulatory RNAs (sRNAs) such as SmsR1532 and SmsR1785 that play important roles in gene regulation . While direct relationships between SMU_988 and these sRNAs are not explicitly described in current literature, contextual evidence suggests:

• SMU_988 may be part of gene regulatory networks influenced by sRNAs

• Its expression might be regulated by SloR, a metal-dependent transcriptional regulator

• The protein may participate in manganese-dependent regulation pathways similar to those involving SmsR1532 and SmsR1785

Understanding these relationships could provide insights into bacterial adaptation mechanisms.

What are the optimal storage and handling conditions for recombinant SMU_988?

For optimal stability and activity of recombinant SMU_988, the following storage and handling conditions are recommended:

The addition of glycerol (recommended final concentration 50%) significantly improves protein stability during freeze-thaw cycles . For experimental manipulations, minimize exposure to room temperature.

What quality control methods should be applied to verify SMU_988 integrity?

To ensure experimental reproducibility, the following quality control methods are recommended:

• SDS-PAGE analysis to confirm protein purity (>90%) and molecular weight (~52-53 kDa for His-tagged protein)

• Western blot analysis using anti-His antibodies to confirm identity

• Mass spectrometry to verify protein integrity and sequence

• Circular dichroism to assess secondary structure integrity

• Dynamic light scattering to evaluate aggregation state

These quality control measures help ensure that experimental results are reliable and reproducible.

What are common technical challenges when working with SMU_988 and how can they be addressed?

Based on experience with similar bacterial recombinant proteins, researchers may encounter these challenges:

• Protein solubility issues: Address by optimizing buffer conditions or adding solubility enhancers like Trehalose (6% recommended)

• Protein aggregation: Minimize by keeping protein concentrated and adding glycerol (50%)

• Limited stability: Aliquot reconstituted protein to avoid freeze-thaw cycles

• Functional assay development: Begin with protein-protein interaction studies if function is unknown

• Structural characterization difficulties: Consider fusion tags or crystallization chaperones

Researchers should document optimization approaches to contribute to the knowledge base for this protein.

How does SMU_988 research contribute to understanding bacterial pathogenicity?

While direct evidence for SMU_988's role in pathogenicity is limited in current literature, research on S. mutans virulence factors provides important context:

• S. mutans pathogenicity is closely linked to metal homeostasis, particularly manganese regulation

• Gene regulatory networks involving SloR and small RNAs contribute to virulence mechanisms

• Understanding uncharacterized proteins like SMU_988 may reveal novel therapeutic targets

Research suggests that proteins regulated in response to environmental conditions (like manganese availability) often contribute to bacterial adaptation and virulence .

What bioinformatic approaches can predict potential functions of SMU_988?

For uncharacterized proteins like SMU_988, the following computational approaches can provide functional insights:

• Protein family classification using tools like PFAM and InterPro

• Structure prediction using AlphaFold or RoseTTAFold

• Protein-protein interaction network prediction

• Genomic context analysis to identify functionally related genes

• Comparative analysis with characterized proteins from related organisms

These approaches can generate testable hypotheses about SMU_988 function when experimental data is limited.

Technical Specifications Table