Recombinant Cyanothece sp. UPF0754 membrane protein PCC7424_0748 (PCC7424_0748)

What is Recombinant Cyanothece sp. UPF0754 membrane protein PCC7424_0748?

Recombinant Cyanothece sp. UPF0754 membrane protein PCC7424_0748 is a protein derived from the cyanobacterium Cyanothece sp. (strain PCC 7424), also known as Synechococcus sp. (strain ATCC 29155). It belongs to the UPF0754 family of membrane proteins, with the full-length protein consisting of 413 amino acids . The protein is available in recombinant form, typically expressed in heterologous systems like E. coli or mammalian cells, and may be tagged with affinity tags such as His-tag to facilitate purification . This protein is part of the membrane proteome of Cyanothece sp., a genus of cyanobacteria known for various biotechnologically important properties including nitrogen fixation and synthesis of diverse bioproducts .

How should researchers store and handle recombinant PCC7424_0748 protein?

Proper storage and handling of recombinant PCC7424_0748 protein is critical for maintaining its stability and biological activity. The protein should be stored at either -20°C or -80°C, with storage conditions impacting shelf life—liquid formulations remain stable for approximately 6 months while lyophilized forms maintain stability for up to 12 months at these temperatures . When working with the protein, researchers should briefly centrifuge vials before opening to ensure contents settle at the bottom . For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, adding glycerol to a final concentration of 5-50% (with 50% being recommended by some manufacturers) before aliquoting for long-term storage . Importantly, repeated freeze-thaw cycles should be strictly avoided as they can compromise protein integrity . For short-term work spanning up to one week, working aliquots can be maintained at 4°C .

What is known about the structural characteristics of PCC7424_0748?

Currently, comprehensive structural data for PCC7424_0748 appears limited in the scientific literature. What we do know is that PCC7424_0748 is classified as a membrane protein belonging to the UPF0754 family . The full-length protein consists of 413 amino acids, suggesting a moderate-sized protein structure . As a membrane protein, it likely contains hydrophobic domains that facilitate integration into cellular membranes, though specific transmembrane domain organization has not been extensively characterized in the available literature. The protein can be expressed with various tags (such as His-tag) for research purposes, which may affect certain structural properties but facilitate purification and downstream applications . Researchers interested in structural studies would need to employ techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy to elucidate the three-dimensional structure, which would significantly advance understanding of this protein's function.

What expression systems are suitable for recombinant PCC7424_0748 production?

The choice of expression system for recombinant PCC7424_0748 production depends on research objectives and downstream applications. According to available data, both prokaryotic (E. coli) and eukaryotic (mammalian cell) expression systems have been successfully employed . E. coli systems offer advantages of rapid growth, high protein yields, and cost-effectiveness, making them suitable for basic biochemical and structural studies . Alternatively, mammalian cell expression systems may provide better post-translational modifications and proper folding for functional studies, though at higher cost and lower yields . When designing expression constructs, researchers should consider incorporating affinity tags (His-tag is commonly used) to facilitate purification, while ensuring these additions don't interfere with the protein's structural integrity or function . For optimal expression, codon optimization based on the host organism may improve yields, particularly for heterologous expression of cyanobacterial proteins that might contain rare codons. Researchers should evaluate expression conditions (temperature, induction time, media composition) through pilot experiments to determine optimal parameters for their specific experimental goals.

How can quasi-experimental designs be applied to study PCC7424_0748 function?

Quasi-experimental designs offer valuable approaches to study PCC7424_0748 function when randomized controlled trials are impractical or ethically challenging. These designs aim to establish cause-and-effect relationships without random assignment of subjects to treatment groups . For PCC7424_0748 research, nonequivalent groups design could be employed by comparing native Cyanothece strains with those where PCC7424_0748 has been knocked down or modified, selecting strains with similar characteristics to minimize confounding variables . Regression discontinuity approaches might be applicable when studying phenotypic effects that manifest at certain threshold levels of protein expression or activity . Natural experiments could be leveraged by studying spontaneous mutations in PCC7424_0748 across different Cyanothece isolates and correlating these with phenotypic differences . To strengthen internal validity, researchers should carefully control for confounding variables such as growth conditions, genetic background, and physiological state of cultures . Combined with molecular and biochemical techniques, quasi-experimental designs can provide robust evidence for protein function while acknowledging limitations in causal inference compared to true experimental designs with randomization.

What purification strategies are most effective for recombinant PCC7424_0748?

Effective purification of recombinant PCC7424_0748 requires careful consideration of its membrane protein nature and expression system characteristics. For His-tagged versions of the protein, immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices represents the primary purification approach . Because PCC7424_0748 is a membrane protein, initial extraction from cell membranes is critical—researchers should evaluate different detergents (e.g., n-dodecyl-β-D-maltoside, CHAPS, Triton X-100) for their effectiveness in solubilizing the protein while maintaining its native conformation. After initial IMAC purification, size exclusion chromatography can remove aggregates and further increase purity. For applications requiring higher purity (>85% as indicated in product specifications), additional chromatographic steps such as ion exchange may be necessary . Quality assessment using SDS-PAGE and Western blotting should confirm identity and purity, while functional assays (if available) can verify that the purified protein retains biological activity. Throughout purification, maintaining protein stability is essential—consider including glycerol, reducing agents, and protease inhibitors in buffers while avoiding conditions that might promote aggregation or denaturation.

What methods can researchers use to characterize the functional properties of PCC7424_0748?

Characterizing the functional properties of PCC7424_0748 requires a multi-faceted approach given the limited existing information about its specific role. Researchers should begin with bioinformatic analyses, employing tools like BLAST, Pfam, and structure prediction algorithms to identify conserved domains and potential functional motifs by comparing PCC7424_0748 with better-characterized membrane proteins . Gene knockout or knockdown studies using CRISPR-Cas9 or RNA interference can reveal phenotypic changes indicating the protein's physiological significance, though these techniques may need optimization for Cyanothece strains . Protein-protein interaction studies utilizing yeast two-hybrid, pull-down assays, or proximity labeling approaches can identify binding partners that provide functional context . Localization studies using fluorescently-tagged versions of PCC7424_0748 can determine precise subcellular positioning, offering clues to function . Biochemical assays testing for specific enzymatic activities should be guided by bioinformatic predictions or observed phenotypes. Additionally, transcriptomic or proteomic profiling comparing wild-type to PCC7424_0748-modified strains can reveal affected pathways and processes. Integration of these diverse approaches is likely necessary to comprehensively characterize this protein's functional properties.

How might PCC7424_0748 be involved in nitrogen fixation or other metabolic pathways?

While direct evidence for PCC7424_0748's involvement in nitrogen fixation or specific metabolic pathways appears limited in the current literature, researchers can explore potential roles through comparative genomic and functional approaches. Cyanothece species are known for their capacity to fix atmospheric nitrogen under anaerobic conditions and utilize alternative nitrogen sources like urea . Examining the regulation of PCC7424_0748 expression under different nitrogen conditions (nitrate, urea, nitrogen starvation, or diazotrophic growth) could reveal nitrogen-responsive patterns suggestive of involvement in nitrogen metabolism . Co-expression analysis identifying genes with similar expression profiles to PCC7424_0748 across conditions may highlight associated pathways. As a membrane protein, PCC7424_0748 might participate in transport processes related to nitrogen compounds, carbon metabolism, or photosynthesis—researchers should consider testing transport capabilities using reconstituted proteoliposomes or membrane vesicles. Metabolomic profiling of PCC7424_0748 mutants could identify accumulated or depleted metabolites pointing to specific pathway involvement. Additionally, examining protein expression under conditions known to activate specific pathways in Cyanothece (such as oxygen-limited conditions that induce nitrogen fixation) may provide functional insights .

What are the challenges in designing genetic manipulation studies for PCC7424_0748?

Designing genetic manipulation studies for PCC7424_0748 presents several significant challenges researchers must address. First, while genetic tools have been developed for Cyanothece PCC 7425, their direct applicability to Cyanothece PCC 7424 (the strain containing PCC7424_0748) may require optimization and validation . Researchers should assess whether RSF1010-derived plasmid vectors successfully used in Cyanothece PCC 7425 and other cyanobacteria function effectively in PCC 7424 . The membrane localization of PCC7424_0748 complicates overexpression studies, as excessive production may disrupt membrane integrity or lead to misfolding and aggregation. For gene knockout or knockdown approaches, researchers must consider potential essentiality—if PCC7424_0748 is essential, complete deletion may be lethal, necessitating conditional systems or partial activity reduction strategies . When designing fluorescent fusion proteins for localization studies, tag placement requires careful consideration to avoid disrupting membrane integration or protein function. Additionally, the polyploidy of cyanobacterial genomes means genetic modifications must achieve complete segregation across all chromosome copies for clear phenotypic analysis. Finally, phenotypic assessment may require specialized conditions or stresses to reveal functional roles, especially if PCC7424_0748 functions under specific environmental conditions or developmental stages.

How can synthetic biology approaches be applied to study PCC7424_0748?

Synthetic biology approaches offer powerful strategies for investigating PCC7424_0748 function and potential applications. Researchers could develop tunable expression systems using well-characterized promoters, allowing precise control over PCC7424_0748 production levels to study dosage effects and avoid toxicity issues common with membrane protein overexpression . Domain swapping experiments, where portions of PCC7424_0748 are exchanged with related proteins of known function, can identify critical functional regions and potentially create chimeric proteins with novel activities. CRISPR interference (CRISPRi) systems adapted for cyanobacteria provide reversible gene repression, enabling study of essential genes and temporal expression control . For protein engineering, directed evolution approaches combining random mutagenesis with selection systems could identify variants with enhanced properties. Researchers might also create synthetic genetic circuits incorporating PCC7424_0748 to study its regulation or exploit its properties in engineered pathways. Importantly, the genetic toolbox developed for Cyanothece PCC 7425, including RSF1010-derived plasmid vectors for promoter analysis and protein overproduction, provides a foundation that may be adaptable to PCC7424_0748 studies . These techniques could ultimately enable applications such as incorporating PCC7424_0748 into engineered strains for biotechnological purposes, similar to how Cyanothece PCC 7425 was engineered to produce the terpene limonene .

What comparative approaches could elucidate PCC7424_0748 function across cyanobacterial species?

Comparative approaches across cyanobacterial species represent a powerful strategy to elucidate PCC7424_0748 function given limited direct experimental data. Researchers should conduct comprehensive phylogenetic analysis of UPF0754 family proteins across diverse cyanobacteria, identifying conserved regions that may indicate functional importance and variable regions potentially responsible for species-specific functions . Genomic context analysis examining neighboring genes across species can reveal functional associations, as genes involved in similar processes often cluster together. Comparing PCC7424_0748 presence/absence patterns with specific metabolic capabilities across cyanobacterial species may highlight correlations with particular pathways or environmental adaptations. Heterologous expression studies introducing PCC7424_0748 into model cyanobacteria like Synechocystis PCC 6803 or Synechococcus PCC 7942 (where genetic tools are well-established) could reveal phenotypic effects and functional insights . Complementation experiments using PCC7424_0748 to rescue phenotypes in mutants of related genes across species may confirm functional conservation. Additionally, comparing expression patterns under standardized conditions (nitrogen limitation, light fluctuations, etc.) across species containing UPF0754 family proteins could identify conserved regulatory patterns suggesting shared functions. These comparative approaches can generate testable hypotheses about PCC7424_0748 function even with limited direct experimental data on the protein itself.

How might post-translational modifications affect PCC7424_0748 function?

Post-translational modifications (PTMs) could significantly impact PCC7424_0748 function, though specific modifications have not been extensively characterized in the available literature. Researchers investigating this aspect should consider that membrane proteins in cyanobacteria may undergo various modifications affecting localization, activity, and interactions. Potential phosphorylation sites could regulate the protein's activity or interactions in response to environmental stimuli, and these sites can be predicted using bioinformatic tools and verified through phosphoproteomics or site-directed mutagenesis studies. Glycosylation, while less common in bacteria than eukaryotes, might occur and affect protein stability or recognition by interaction partners. For experimental verification, researchers could employ mass spectrometry-based approaches to identify PTMs on purified native PCC7424_0748 from Cyanothece cultures grown under different conditions (varying light, nitrogen sources, or stress conditions) . Comparing PTM patterns across growth conditions could reveal regulatory mechanisms. Additionally, creating site-directed mutants that mimic or prevent specific modifications (phosphomimetic or phosphonull mutations) would allow functional testing of their importance. When expressing recombinant PCC7424_0748, researchers should consider whether the chosen expression system (E. coli vs. mammalian cells) can reproduce relevant PTMs, as this may affect functional studies .

What role might PCC7424_0748 play in Cyanothece's ability to grow on alternative nitrogen sources?

PCC7424_0748's potential role in Cyanothece's ability to utilize alternative nitrogen sources presents an intriguing research direction. Cyanothece species demonstrate remarkable metabolic versatility, including the capacity to grow on urea as the sole nitrogen source (up to 2 mM concentration) and fix atmospheric nitrogen under anaerobic conditions . As a membrane protein, PCC7424_0748 could potentially function in nitrogen compound transport, sensing, or signaling processes. Researchers should investigate whether PCC7424_0748 expression levels change when cells are grown on different nitrogen sources (nitrate, urea, ammonium, or diazotrophic conditions), which would suggest involvement in nitrogen metabolism regulation . Genetic modification approaches (knockdown, overexpression, or site-directed mutagenesis) followed by growth assessment on various nitrogen sources could directly test this protein's importance in nitrogen utilization. Protein localization studies might reveal whether PCC7424_0748 positions near known nitrogen metabolism machinery or transporters. Interaction studies could identify whether it associates with characterized proteins involved in urea uptake, processing, or nitrogen fixation machinery. Additionally, comparative genomic analysis across Cyanothece strains with differing nitrogen utilization capabilities might reveal correlations with PCC7424_0748 sequence variations. Understanding this protein's role in nitrogen metabolism could have significant implications for both basic cyanobacterial physiology and applications in bioremediation or sustainable agriculture.

How could PCC7424_0748 be utilized in biotechnological applications?

PCC7424_0748's potential in biotechnological applications remains largely unexplored but offers several promising avenues based on Cyanothece characteristics. If functional studies reveal involvement in nitrogen metabolism or urea utilization, engineered strains with modified PCC7424_0748 expression could enhance bioremediation of urea-contaminated waters, effectively coupling water treatment with valuable biomass production . As a membrane protein, PCC7424_0748 might serve as a component in biosensor development, potentially creating systems that respond to specific environmental conditions or compounds. Researchers could explore incorporating PCC7424_0748 into synthetic biology platforms similar to those developed for Cyanothece PCC 7425, which has been engineered to produce high-value compounds like limonene (a terpene with applications in biofuels, bioplastics, cosmetics, food, and pharmaceuticals) . If structural studies reveal unique properties, the protein could serve as a scaffold for designing novel membrane protein-based technologies. Additionally, understanding PCC7424_0748's role might contribute to engineering more robust photosynthetic systems with enhanced carbon fixation or improved tolerance to environmental stresses. These applications would require comprehensive characterization of the protein's function and development of reliable genetic manipulation systems specifically optimized for Cyanothece PCC 7424, building upon the genetic toolbox established for related strain PCC 7425 .

What research gaps need to be addressed to fully understand PCC7424_0748 function?

Several critical research gaps must be addressed to fully understand PCC7424_0748 function. Foremost, basic characterization data appears lacking in the literature—researchers should prioritize determining the protein's precise subcellular localization, membrane topology, and three-dimensional structure through techniques like fluorescent tagging, protease accessibility assays, and structural biology approaches . Gene knockout or knockdown studies in Cyanothece PCC 7424 would provide fundamental insights into whether the protein is essential and what phenotypic consequences result from its absence or reduction. Comprehensive interactome analysis identifying binding partners through techniques like co-immunoprecipitation followed by mass spectrometry could reveal functional associations . Transcriptomic and proteomic profiling comparing wild-type to PCC7424_0748-modified strains across various growth conditions would highlight affected pathways. Development of functional assays specific to hypothesized activities (transport, sensing, enzymatic) represents another critical gap. Additionally, the regulatory mechanisms controlling PCC7424_0748 expression remain unexplored—characterizing its promoter and identifying regulatory factors would provide contextual understanding. Establishing reliable genetic manipulation systems specifically for Cyanothece PCC 7424, adapting tools developed for PCC 7425, would enable many of these studies . Finally, comparative studies examining homologs across diverse cyanobacteria could provide evolutionary context and functional insights. Addressing these gaps would provide a comprehensive understanding of this currently enigmatic membrane protein.