Recombinant Nautilus macromphalus Uncharacterized protein IMPP7

What is Nautilus macromphalus and why are its uncharacterized proteins significant for research?

Nautilus macromphalus, commonly known as the Bellybutton nautilus, represents one of the most ancient lineages of cephalopod mollusks. As a "living fossil," this organism provides unique insights into evolutionary biology and molecular adaptations . Uncharacterized proteins from N. macromphalus, including IMPP7, are significant because they may represent molecular mechanisms conserved over hundreds of millions of years of evolution. These proteins could be involved in specialized physiological processes related to deep-sea adaptation, shell formation, or unique metabolic pathways that have allowed nautiloids to persist largely unchanged through geological time while other cephalopod lineages have diversified extensively .

What are the current storage and handling recommendations for recombinant IMPP7?

Based on established protocols for similar uncharacterized proteins from Nautilus macromphalus, the following storage and handling guidelines are recommended:

• Store at -20°C for regular use, or -80°C for extended storage periods

• Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

• Prior to opening, briefly centrifuge the vial to bring contents to the bottom

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

• For long-term storage, add glycerol to a final concentration of 5-50% (typically 50%)

The shelf life of reconstituted protein is generally 6 months at -20°C/-80°C in liquid form, while lyophilized preparations can maintain stability for up to 12 months when stored at -20°C/-80°C .

What expression systems are available for producing recombinant Nautilus macromphalus proteins?

Multiple expression systems are available for the production of recombinant N. macromphalus proteins, each with distinct advantages for different research applications:

The choice of expression system should be guided by the specific experimental goals, predicted protein characteristics, and intended downstream applications .

What purification approaches are most effective for recombinant proteins from ancient marine organisms?

Purification of recombinant proteins from ancient marine organisms like Nautilus presents unique challenges that require adaptations to standard protocols:

• Initial extraction considerations: When working with tagged recombinant proteins, select appropriate affinity chromatography methods based on the tag (His-tag, Avi-tag biotinylated proteins, etc.)

• Additional purification steps: Following initial purification, size exclusion chromatography is often necessary to achieve >85% purity (standard for commercially available recombinant Nautilus proteins)

• Stability optimization: Buffer conditions may need optimization to maintain stability of proteins from deep-sea organisms that evolved under high pressure conditions (N. macromphalus inhabits depths of approximately 400m)

• Quality control assessment: SDS-PAGE verification is standard for confirming purity (>85% is typically considered acceptable for research applications)

• Protein-specific adaptations: The small size of many uncharacterized Nautilus proteins (similar proteins like IMPP20 consist of only 14 amino acids) may require specialized approaches such as tricine gels rather than standard SDS-PAGE

What approaches can be used to determine the biological function of uncharacterized proteins like IMPP7?

Determining the biological function of uncharacterized proteins from Nautilus macromphalus requires a multi-faceted approach:

• Bioinformatic prediction: Though limited by the evolutionary distance between Nautilus and model organisms, sequence analysis and structural predictions may provide initial functional hypotheses. The complete mitochondrial genome sequencing of N. macromphalus and comparison with Allonautilus (approximately 8.3% sequence divergence) offers some context for evolutionary relationships .

• Localization studies: Determine where IMPP7 is expressed in Nautilus tissues. Particularly noteworthy would be expression in specialized structures like the pericardial appendages, which harbor unique bacterial symbionts in a dual symbiotic relationship .

• Interaction studies: Identify binding partners through co-immunoprecipitation or pull-down assays. The biotinylated Avi-tag version of the protein (CSB-EP******NBA-B) is particularly suited for such studies .

• Physiological context exploration: Consider potential roles in unique physiological processes of Nautilus, such as:

  • Shell formation and biomineralization

  • Adaptation to deep-sea pressure conditions

  • Specialized excretory functions (N. macromphalus has a unique excretory system with bacterial symbionts producing ammonia-rich excretory fluid)

  • Buoyancy regulation mechanisms

• Comparative functional studies: Examine functional differences between homologous proteins in Nautilus and other cephalopods to identify specialized adaptations.

How can recombinant IMPP7 be used to study evolutionary relationships among cephalopods?

Recombinant IMPP7 provides several opportunities for evolutionary studies among cephalopods:

How might IMPP7 relate to the unique symbiotic relationships found in Nautilus macromphalus?

N. macromphalus exhibits a remarkable dual bacterial symbiosis in its pericardial appendage (excretory organ) that could potentially involve uncharacterized proteins like IMPP7:

• Symbiont relationship: The excretory organ harbors a high density of two phylogenetically distinct bacterial types - a β-proteobacterium (related to ammonia-oxidizing bacteria, ≥87.5% sequence similarity) and a coccoid spirochaete (related to marine Spirochaeta species, ≥89.8% sequence similarity) .

• Potential roles for IMPP7:

  • Mediating host-symbiont recognition

  • Regulating symbiont populations

  • Facilitating metabolite exchange between host and symbionts

  • Supporting the excretory process that produces ammonia-rich fluid

• Experimental approaches: To investigate potential roles of IMPP7 in this symbiosis, researchers could:

  • Use fluorescently labeled recombinant IMPP7 to examine binding with bacterial symbionts

  • Develop antibodies against IMPP7 to determine its localization relative to the baso-medial region of pericardial villi where symbionts occur

  • Test for interactions between IMPP7 and bacterial surface proteins

• Evolutionary significance: This symbiotic relationship may represent an ancient adaptation, and proteins involved could provide insights into the evolution of host-microbe interactions in marine environments .

What insights can isotope studies combined with protein analysis provide about the habitat and physiology of Nautilus macromphalus?

Integrating isotopic data with protein studies of N. macromphalus offers a powerful approach to understanding this organism's ecology and physiology:

• Habitat depth characterization: Isotopic evidence from oxygen and carbon isotopes (δ18O, δ13C) in nautilus shells provides insights into habitat depth through ontogeny. Nautilus macromphalus specimens have been captured at depths of approximately 400m .

• Metabolic carbon analysis: Studies have investigated the fraction of metabolic carbon in nautilus shells. Proteins involved in shell formation or metabolic regulation, potentially including uncharacterized proteins like IMPP7, may show adaptations related to these specialized processes .

• Ontogenetic changes: Researchers have examined whether morphological changes during ontogeny are reflected in isotopic values of shells. Similarly, the expression and function of proteins like IMPP7 may vary through developmental stages .

• Research integration approaches:

  • Correlate protein expression patterns with isotopic signatures across developmental stages

  • Examine whether proteins like IMPP7 show structural adaptations consistent with the environmental conditions indicated by isotopic data

  • Investigate potential roles of uncharacterized proteins in biomineralization processes that would affect isotopic incorporation

• Methodological considerations: When designing experiments to investigate these relationships, researchers should consider both spatial localization of proteins within tissues and temporal expression patterns throughout development .

What quality control measures should be implemented when working with recombinant Nautilus proteins?

Comprehensive quality control for recombinant Nautilus proteins should include:

• Purity assessment:

  • SDS-PAGE analysis with target purity >85% as standard for research applications

  • Mass spectrometry verification of protein identity and detection of potential contaminants

• Structural integrity verification:

  • Circular dichroism to assess secondary structure

  • Size exclusion chromatography to confirm proper oligomeric state

  • Thermal shift assays to evaluate stability under experimental conditions

• Functional verification (where possible):

  • Activity assays if function can be predicted

  • Binding studies with potential interaction partners

  • Comparison of properties across different expression systems

• Batch consistency monitoring:

  • Maintaining consistent expression and purification protocols

  • Comparing new batches to reference standards

  • Documenting lot-to-lot variation in critical parameters

• Storage stability testing:

  • Monitoring activity/integrity over time under recommended storage conditions

  • Establishing maximum freeze-thaw cycles before detectable degradation occurs

How can researchers address the challenges of working with proteins from deep-sea organisms in standard laboratory conditions?

Working with proteins from deep-sea organisms like Nautilus macromphalus (found at depths of approximately 400m) presents unique challenges that require specialized approaches:

• Pressure considerations:

  • Proteins evolved under high hydrostatic pressure may have different structural stability at atmospheric pressure

  • Consider using high-pressure chambers for functional assays to better approximate native conditions

  • Compare activity/structure under varied pressure conditions to assess pressure adaptation effects

• Temperature adaptations:

  • Deep-sea environments have stable, often colder temperatures than surface waters

  • Optimize assay temperatures based on the thermal environment of Nautilus habitat

  • Assess thermal stability and activity profiles across a range of temperatures

• Oxidation sensitivity:

  • Proteins from low-oxygen environments may be more sensitive to oxidation

  • Consider including reducing agents in buffers

  • Minimize exposure to air during handling and storage

• Buffer optimization:

  • Deep-sea organisms experience different ionic conditions than surface organisms

  • Test protein stability and activity in buffers mimicking the ionic composition of deep-sea environments

  • Consider the effect of pressure on pH in buffer design

• Reference materials:

  • When possible, compare recombinant protein properties with native protein extracted directly from Nautilus tissues

  • Use multiple expression systems to identify potential artifacts introduced by the recombinant expression process

How does the mitochondrial genome information from Nautilus macromphalus inform studies of its uncharacterized proteins?

The complete mitochondrial genome sequence of Nautilus macromphalus provides valuable context for studies of its uncharacterized proteins:

• Evolutionary rate calibration: The mitogenome comparison between N. macromphalus and Allonautilus scrobiculatus revealed varying rates of evolution across different genes (from identical tRNA sequences to >15% divergence in ATP8). This variation provides a framework for understanding evolutionary pressures on different proteins .

• Gene arrangement insights: The gene order in the N. macromphalus mitogenome is identical to that found in Allonautilus, suggesting evolutionary stability in this ancient lineage. This genomic stability may extend to nuclear-encoded proteins like IMPP7 .

• Base composition patterns: Analysis of base composition heterogeneity around the mitochondrial genome can inform codon optimization strategies for recombinant expression of Nautilus proteins .

• Noncoding region variation: The largest noncoding region in the Nautilus mitogenome contains a 62 bp repeat that varies in copy number between species. Similar repetitive elements might be found in the regulatory regions of nuclear genes encoding proteins like IMPP7 .

• Taxonomic context: Mitochondrial genome data has contributed to understanding the phylogenetic relationships among nautiloids, including the identification of new species. This taxonomic framework is essential for comparative studies of proteins across nautiloid species .

What emerging technologies could advance our understanding of uncharacterized proteins from ancient marine lineages?

Several cutting-edge technologies hold promise for advancing our understanding of uncharacterized proteins from ancient marine lineages like Nautilus:

• AlphaFold and other AI structure prediction tools:

  • Can predict structures of uncharacterized proteins with increasing accuracy

  • Particularly valuable for ancient lineages where experimental structures may be difficult to obtain

  • Could help identify potential functional sites and interaction surfaces

• Single-cell transcriptomics and proteomics:

  • Allow identification of cell types expressing specific proteins

  • Can reveal developmental and tissue-specific expression patterns

  • Particularly valuable for understanding spatiotemporal expression of proteins like IMPP7

• High-pressure structural biology techniques:

  • High-pressure NMR and X-ray crystallography

  • Cryo-EM under simulated pressure conditions

  • Enable structural characterization under conditions more similar to native deep-sea environment

• Advanced mass spectrometry approaches:

  • Cross-linking mass spectrometry to identify interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to probe structural dynamics

  • Native mass spectrometry to determine oligomeric states under near-native conditions

• CRISPR/Cas9-based functional genomics:

  • While challenging to apply directly to Nautilus, could be used in model systems to study homologs

  • Potential for development of cell culture systems from nautiloid tissues

  • Creation of heterologous expression systems for functional testing

• Environmental metagenomics/metaproteomics:

  • Study nautiloid-associated microbiomes in greater depth

  • Examine potential roles of proteins like IMPP7 in host-microbe interactions

  • Place protein function in ecological context

These emerging technologies, when applied to uncharacterized proteins from ancient marine lineages, promise to bridge the gap between sequence data and functional understanding, potentially revealing molecular mechanisms conserved over hundreds of millions of years of evolution.