Recombinant Nitrosomonas europaea UPF0301 protein NE1668 (NE1668)

What is UPF0301 protein NE1668 and what organism does it originate from?

UPF0301 protein NE1668 is a protein found in Nitrosomonas europaea, a chemolithotrophic bacterium that obtains energy through ammonia oxidation and fixes carbon from carbon dioxide. The UPF designation (Uncharacterized Protein Family) indicates that the protein's function has not been fully elucidated. As a member of the UPF0301 family, it likely has conserved structural features common to this protein group, though its specific role in N. europaea remains an area of active investigation .

Why is Nitrosomonas europaea significant for protein research?

Nitrosomonas europaea represents a valuable model organism for studying specialized metabolic processes. This bacterium is notable for its chemolithotrophic metabolism, obtaining energy through ammonia oxidation to hydroxylamine (catalyzed by ammonia monooxygenase) and further to nitrite (catalyzed by hydroxylamine dehydrogenase). The organism also fixes carbon dioxide via the Calvin-Benson-Bassham cycle, utilizing RubisCO . Additionally, N. europaea possesses multiple toxin-antitoxin systems believed to be associated with its slow growth rate and stress adaptation mechanisms, making it particularly interesting for studying protein-mediated cellular responses to environmental challenges .

What expression systems are recommended for recombinant production of NE1668?

For recombinant production of UPF0301 protein NE1668, multiple expression hosts can be utilized with varying advantages:

E. coli and yeast systems offer the best yields and shorter turnaround times, making them preferred for initial studies and applications not requiring extensive post-translational modifications. For studies where protein folding or activity depends on specific modifications, insect or mammalian cell expression systems are recommended despite their longer production times .

What optimization strategies are recommended for maximizing soluble NE1668 expression in E. coli?

Optimizing soluble expression of recombinant proteins like NE1668 in E. coli requires systematic evaluation of multiple variables. Based on experimental design approaches used for similar proteins, researchers should consider:

• Induction parameters: Optimal results often occur with moderate IPTG concentrations (0.1-0.5 mM) and induction at mid-log phase (OD600 of 0.6-0.8)

• Temperature: Lower temperatures (15-25°C) typically favor soluble expression by slowing protein synthesis and allowing proper folding

• Media composition: Enhanced yields can be achieved using media with balanced nitrogen sources (e.g., 5 g/L yeast extract, 5 g/L tryptone) and moderate salt concentrations (10 g/L NaCl)

• Carbon source: Low glucose concentrations (0.5-1 g/L) can help regulate expression rates

• Duration: Extended expression periods (4-16 hours) at lower temperatures often maximize soluble protein yields

Statistical experimental design approaches, such as factorial designs, allow for efficient optimization by testing multiple variables simultaneously. This methodology has demonstrated success in achieving high-level expression (>200 mg/L) of soluble, functional recombinant proteins from various bacterial sources .

How can researchers evaluate the proper folding and activity of recombinant NE1668?

Evaluating proper folding and activity of recombinant NE1668 requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure elements

  • Thermal shift assays to evaluate protein stability

  • Size exclusion chromatography to confirm monomeric state or expected oligomerization

• Functional characterization:

  • While the specific function of UPF0301 remains uncharacterized, binding assays with potential interacting partners from N. europaea should be conducted

  • For proteins from the same organism, specific activity assays have been developed (e.g., endoribonuclease activity for MazF proteins that cleave at specific sequence motifs)

• Homogeneity assessment:

  • SDS-PAGE analysis (target >75% homogeneity)

  • Western blotting with anti-His or other epitope-specific antibodies if tagged versions are used

What is known about the structural features of UPF0301 family proteins?

While specific structural data for NE1668 is limited, inferring from related UPF0301 family proteins:

• These proteins typically feature conserved structural domains that may include distinctive secondary structure elements

• Sequence analysis suggests potential binding sites that could interact with nucleic acids or other proteins

• Comparative analysis with characterized members of the UPF0301 family can provide insight into potential structural motifs

Using bioinformatics approaches to align NE1668 with characterized UPF proteins may reveal conserved regions that indicate functional domains. Researchers should consider performing structural prediction using tools like AlphaFold2 to generate hypotheses about functional regions prior to experimental verification .

How does the genomic context of NE1668 inform potential functional roles?

The genomic context analysis of NE1668 within the N. europaea genome can provide valuable insights into its potential function:

• Examine neighboring genes for functional relationships (operonic structure)

• Analyze the presence of regulatory elements in the promoter region

• Compare synteny across related organisms to identify conserved genetic neighborhoods

N. europaea possesses multiple toxin-antitoxin systems, including five MazEF loci, which play roles in stress adaptation. This genomic context suggests potential involvement in stress response mechanisms. The analysis of triplet sequences (e.g., TGG motifs recognized by MazF endoribonucleases) in the NE1668 transcript could indicate whether it might be regulated by these systems under stress conditions .

How might NE1668 be involved in N. europaea's adaptation to environmental stresses?

N. europaea utilizes multiple stress-response mechanisms, with toxin-antitoxin systems playing a particularly important role. While the specific function of NE1668 remains to be characterized, several hypotheses warrant investigation:

• Potential role in metabolic regulation during stress conditions, particularly related to ammonia oxidation or carbon fixation pathways

• Possible involvement in dormancy or viable but non-culturable (VBNC) states known to improve bacterial stress resistance

• Regulation by or interaction with the MazF endoribonuclease system, which selectively targets transcripts containing UGG motifs

Statistical analysis of other N. europaea transcripts has revealed that genes essential for core metabolic functions (e.g., hydroxylamine dehydrogenase for ammonia oxidation and RubisCO for carbon fixation) are particularly enriched in MazF recognition sites, suggesting a regulatory mechanism that would quickly shut down energy-intensive processes during stress. Examining NE1668 for similar regulatory patterns could provide insight into its functional importance .

What methodologies are recommended for investigating protein-protein interactions involving NE1668?

To elucidate the potential interactions of NE1668 with other proteins in N. europaea, researchers should consider a multi-faceted approach:

• Co-immunoprecipitation (Co-IP) using tagged versions of NE1668 expressed in homologous or heterologous systems

• Bacterial two-hybrid screening to identify interacting partners

• Pull-down assays with recombinant NE1668 using N. europaea cell lysates

• Cross-linking mass spectrometry (XL-MS) to capture transient protein interactions

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding kinetics with candidate interacting partners

When investigating potential toxin-antitoxin relationships, researchers should examine whether NE1668 expression causes growth inhibition that can be counteracted by co-expression of a putative antitoxin partner, similar to studies conducted with MazF and its cognate MazE antitoxin in N. europaea .

What strategies can address low solubility issues when expressing NE1668?

When encountering solubility challenges with recombinant NE1668, consider implementing these targeted approaches:

• Expression optimization:

  • Reduce expression temperature to 15-20°C

  • Lower inducer concentration (0.01-0.05 mM IPTG)

  • Use auto-induction media for gradual protein expression

• Fusion tags and partners:

  • Test multiple solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Position tags at N-terminus or C-terminus to determine optimal configuration

• Buffer optimization:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test additives such as arginine (50-500 mM), low concentrations of non-ionic detergents, or osmolytes like glycerol (5-20%)

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • If NE1668 functions as part of a complex, co-express with potential binding partners from N. europaea

How can researchers distinguish between functional and non-functional recombinant NE1668 preparations?

Distinguishing functional from non-functional recombinant protein preparations requires comprehensive quality assessment:

• Biophysical characterization:

  • Differential scanning fluorimetry to assess thermal stability profiles

  • Dynamic light scattering to detect aggregation

  • Native PAGE to evaluate conformational homogeneity

• Functional validation (when specific activity is unknown):

  • Ability to interact with known binding partners from N. europaea

  • Conservation of predicted structural elements via circular dichroism

  • Comparative analysis with wild-type protein extracted from native source

• Post-translational modification analysis:

  • Mass spectrometry to identify modifications present in native vs. recombinant protein

  • Phosphorylation, acetylation, or other modifications may be critical for function

• Active site integrity:

  • If binding sites or catalytic residues are predicted, site-directed mutagenesis followed by structural and limited functional analysis can validate proper folding around critical regions