Recombinant Spiroplasma virus SpV1-C74 Uncharacterized protein ORF15 (ORF15)

What is the Spiroplasma virus SpV1-C74 Uncharacterized protein ORF15?

Spiroplasma virus SpV1-C74 Uncharacterized protein ORF15 is a 72-amino acid viral protein (UniProt ID: Q88417) encoded by the ORF15 gene of Spiroplasma virus SpV1-C74. The full amino acid sequence is: MKIKILKFLKRKKWWEILVYILVVGFGFALFIGSIYDKWDKLIKWERYFILIYVSCKFVFLIWISLMYFIYN . This protein is classified as "uncharacterized" because its precise biological function remains to be fully elucidated through structural and functional studies. For recombinant expression, it is typically produced with an N-terminal His-tag to facilitate purification and is expressed in E. coli expression systems .

What structural characteristics can be inferred from the ORF15 protein sequence?

Analysis of the ORF15 amino acid sequence reveals several notable structural features:

What expression and purification protocols are recommended for recombinant ORF15 protein?

For optimal expression and purification of recombinant ORF15 protein, researchers should follow this methodological approach:

• Expression system: Use E. coli as the heterologous expression host with an N-terminal His-tag fusion construct .

• Purification protocol:

  • Employ immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

  • Perform elution with imidazole gradient

  • Achieve >90% purity as verified by SDS-PAGE

• Post-purification processing:

  • Supply as lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol (recommended final concentration 50%) for long-term storage

This methodology consistently yields high-purity recombinant protein suitable for downstream applications in research settings.

What storage conditions maintain optimal stability of purified ORF15 protein?

The stability of recombinant ORF15 protein is highly dependent on proper storage conditions. Based on empirical data, the following protocol is recommended:

• Short-term storage: Store working aliquots at 4°C for up to one week to minimize freeze-thaw cycles .

• Long-term storage:

  • Store at -20°C/-80°C upon receipt

  • Aliquot to prevent repeated freeze-thaw cycles

  • Add glycerol (5-50%, with 50% being optimal) as a cryoprotectant

• Reconstitution guidelines:

  • Briefly centrifuge vial before opening

  • Reconstitute in deionized sterile water

  • After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

These storage recommendations are designed to preserve protein integrity and functionality for extended periods, which is critical for experimental reproducibility.

How can structural biology approaches be applied to characterize ORF15?

Structural characterization of ORF15 requires a multi-faceted approach due to its small size (72 amino acids) and potential membrane association:

The comprehensive structural information obtained through these methods would provide critical insights into potential functional domains and interaction interfaces of ORF15 .

What functional genomics approaches can elucidate the role of ORF15 in viral biology?

To investigate the biological function of the uncharacterized ORF15 protein, researchers should implement a systematic functional genomics strategy:

• Comparative genomics analysis:

  • Align ORF15 sequences across different Spiroplasma virus strains

  • Identify conserved domains suggesting functional importance

  • Map sequence variations to potential phenotypic differences

• Protein-protein interaction studies:

  • Perform pull-down assays using His-tagged ORF15 as bait

  • Identify host bacterial proteins that interact with ORF15

  • Validate interactions using complementary techniques (co-immunoprecipitation, FRET)

• Genetic manipulation approaches:

  • Generate ORF15 knockout or mutant viruses if feasible

  • Assess effects on viral replication, assembly, and host interaction

  • Conduct complementation studies to confirm phenotypes

These methodological approaches provide complementary data to construct a comprehensive understanding of ORF15's role in viral biology .

What are common challenges in ORF15 expression and purification, and how can they be addressed?

Researchers working with recombinant ORF15 protein may encounter several technical challenges:

Systematic optimization of expression and purification parameters is essential to overcome these challenges and obtain high-quality protein for downstream applications .

How can researchers validate the quality and functionality of purified ORF15 protein?

Quality assessment of purified ORF15 protein should include multiple analytical techniques:

• Purity assessment:

  • SDS-PAGE analysis (target: >90% purity)

  • Size exclusion chromatography to detect aggregation

  • Mass spectrometry to confirm molecular weight (expected: approximately 8-9 kDa plus tag)

• Structural integrity validation:

  • Circular dichroism to confirm proper secondary structure formation

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to assess compact folding

• Functional validation:

  • Binding assays with potential interaction partners

  • Lipid interaction studies if membrane association is suspected

  • Activity assays based on bioinformatic function predictions

This multi-parameter approach ensures that the recombinant protein maintains native-like properties suitable for reliable experimental results .

How should researchers design experiments to investigate ORF15-host membrane interactions?

Based on sequence analysis suggesting potential membrane association, researchers investigating ORF15-membrane interactions should consider this methodological framework:

• In vitro membrane binding assays:

  • Prepare liposomes with compositions mimicking Spiroplasma membranes

  • Incubate purified ORF15 with liposomes and assess binding through co-sedimentation

  • Analyze binding specificity by varying lipid compositions

• Biophysical characterization:

  • Conduct surface plasmon resonance (SPR) with immobilized lipid bilayers

  • Perform Förster resonance energy transfer (FRET) between labeled ORF15 and membrane probes

  • Use atomic force microscopy to visualize membrane associations

• Structural studies in membrane mimetics:

  • Solve protein structure in detergent micelles or nanodiscs

  • Identify membrane-interacting residues through hydrogen-deuterium exchange

These complementary approaches would provide mechanistic insights into how ORF15 may interact with host cell membranes during viral infection .

What experimental approaches can resolve contradictory findings in ORF15 research?

When researchers encounter contradictory results in ORF15 functional studies, they should implement a systematic troubleshooting approach:

• Standardize experimental conditions:

  • Use consistent protein preparation methods

  • Control buffer conditions, temperature, and incubation times

  • Document all experimental parameters meticulously

• Employ orthogonal techniques:

  • Validate key findings using multiple independent methods

  • Compare results from in vitro and in vivo systems

  • Use both structural and functional approaches

• Isolate variables systematically:

  • Test effect of tags and fusion partners

  • Evaluate concentration-dependent effects

  • Assess impacts of experimental buffers and additives

• Collaborative validation:

  • Engage independent laboratories to replicate critical experiments

  • Use standardized protocols and reagents

  • Report both positive and negative results

This methodological approach helps identify sources of experimental variation and builds consensus around reproducible findings .