Recombinant UPF0303 protein CTC_00911 (CTC_00911)

What is known about the genomic context of CTC_00911?

CTC_00911 is positioned within an operon (CTC00903 to CTC00911) in the Clostridium tetani genome that appears to be involved in carbohydrate metabolism and transport. This operon includes genes encoding an rbsD-fucU mutarotase, a LacI family transcriptional regulator, a d-ribose transporter ATP-binding protein, a ribose ABC transporter permease, a ribose ABC transporter substrate-binding protein, a d-Arabinose-5-Phosphate Isomerase (CtAPI), a ribokinase, a d-glucose:d-fructose oxidoreductase, and finally CTC_00911 itself, which is annotated as a hypothetical protein . The presence of CTC_00911 in this carbohydrate metabolism operon suggests it may play a role in sugar processing or transport, though its specific function remains uncharacterized. Understanding this genomic neighborhood provides important contextual clues for designing functional studies.

Which expression system should I use for recombinant CTC_00911 production?

While traditional E. coli expression systems remain viable options, Vibrio natriegens has emerged as a promising alternative expression host that may offer specific advantages for producing CTC_00911. V. natriegens has shown superior protein folding capabilities for several challenging proteins that were difficult to produce in E. coli . For CTC_00911 expression, consider the following approach:

• Attempt parallel expression in both E. coli and V. natriegens expression systems

• For E. coli expression, test multiple conditions including:

  • LB medium at 37°C

  • Dynamite medium at 16°C

  • Autoinduction (ZYM) medium at 20°C

• For V. natriegens, utilize:

  • Brain Heart Infusion medium supplemented with NaCl at 30°C

  • Shorter induction times (4-5 hours) compared to E. coli

Recent studies have demonstrated that V. natriegens can produce higher yields of properly folded protein for certain targets, with increases of 40% or more compared to E. coli . Additionally, the absence of ArnA contaminant in V. natriegens may result in higher purity during initial IMAC purification steps .

What purification strategies are recommended for CTC_00911?

Based on successful approaches with other proteins from Gram-positive bacteria, a multi-step purification strategy is recommended:

• Initial capture: Utilize immobilized metal affinity chromatography (IMAC) with an N-terminal His-tag. This approach has proven effective for purification of other proteins from C. tetani, such as CtAPI, achieving >95% purity in a single step .

• Tag removal: Incorporate a TEV protease cleavage site between the His-tag and CTC_00911. Optimize cleavage conditions, as TEV protease efficiency can vary significantly between expression systems. For instance, proteins expressed in V. natriegens often demonstrate better TEV cleavage compared to the same proteins from E. coli, suggesting improved protein folding .

• Polishing step: Employ size exclusion chromatography (SEC) to:

  • Remove aggregates

  • Determine the oligomeric state of CTC_00911

  • Assess protein quality

For example, the related protein CtAPI was found to form tetramers with an apparent molecular mass of 109.2 kDa (4.26 times the calculated subunit mass) using gel filtration chromatography . Determining the oligomeric state of CTC_00911 may provide functional insights.

How can I improve solubility if CTC_00911 forms inclusion bodies?

If CTC_00911 forms inclusion bodies, several strategies can be implemented:

• Utilize solubility-enhancing fusion partners:

  • MBP (Maltose Binding Protein) fusion has shown success with other challenging proteins in both E. coli and V. natriegens

  • Test multiple fusion partners in parallel (MBP, SUMO, Thioredoxin)

• Optimize expression conditions:

  • Lower induction temperature (though note that V. natriegens typically performs better at 30°C rather than lower temperatures)

  • Reduce inducer concentration

  • Evaluate co-expression with chaperones

• Consider V. natriegens as an alternative host:

  • Several proteins, including nanobodies and kinase domains, show improved folding in V. natriegens compared to E. coli

  • The rapid growth rate of V. natriegens (doubling time ≤10 min) allows for faster optimization cycles

• Media optimization:

  • Supplement with cofactors potentially important for folding (e.g., zinc for cysteine-rich domains)

  • Test auto-induction media formulations

What initial assays should be performed to begin characterizing CTC_00911?

To begin functional characterization of CTC_00911, the following analytical approaches are recommended:

• Biophysical characterization:

  • Differential Scanning Fluorimetry (DSF) to determine thermal stability (Tm)

  • Circular Dichroism (CD) to assess secondary structure

  • Dynamic Light Scattering (DLS) to evaluate homogeneity

• Activity screening based on genomic context:

  • Test for enzymatic activities related to carbohydrate metabolism

  • Screen for interactions with other proteins in the operon, particularly CtAPI

  • Assess binding to various sugar substrates

• Structural analysis:

  • Crystallization trials

  • NMR spectroscopy for solution structure (requires isotopic labeling)

For isotopic labeling, adapt the protocol used for other proteins in V. natriegens: culture in ModM9 medium amended with 4 g/L glucose, 15 g/L NaCl, and 50 mM 15NH4Cl, induce with 1 mM IPTG at OD600 = 0.5, and incubate at 25°C overnight .

How might the function of CTC_00911 be related to d-Arabinose-5-Phosphate metabolism?

Given CTC_00911's position in an operon containing d-Arabinose-5-Phosphate Isomerase (CtAPI), investigating potential functional relationships is warranted. Consider these experimental approaches:

• Metabolic profiling:

  • Compare metabolite profiles between wild-type C. tetani and CTC_00911 knockout strains

  • Focus on carbohydrate intermediates, particularly pentose phosphate pathway metabolites

  • Quantify d-Arabinose-5-Phosphate (A5P) and ribulose-5-phosphate (Ru5P) levels

• Protein-protein interaction studies:

  • Perform pull-down assays with tagged CTC_00911 to identify binding partners

  • Use bacterial two-hybrid systems to test for direct interaction with CtAPI

  • Employ crosslinking strategies followed by mass spectrometry

• Enzymatic activity screening:

  • Test CTC_00911 for complementary enzymatic activities to CtAPI

  • Investigate potential roles in regulating A5P/Ru5P interconversion

  • Assess activity with various sugar phosphates as substrates

While CtAPI specifically catalyzes the interconversion of A5P and Ru5P , CTC_00911 might be involved in regulating this isomerization, similar to how some Gram-negative bacteria use A5P metabolism for regulating cellular d-glucitol uptake .

What advanced structural biology approaches are most suitable for CTC_00911 characterization?

For comprehensive structural characterization of CTC_00911, implement the following advanced approaches:

• X-ray crystallography optimization:

  • Screen multiple constructs with varied N- and C-terminal boundaries

  • Test crystallization with binding partners or substrates

  • Consider surface entropy reduction mutations to promote crystal contacts

• Solution NMR studies:

  • Produce 2H/13C/15N-labeled CTC_00911 in V. natriegens

  • For deuteration in V. natriegens, adapt protocols developed for other challenging proteins

  • Collect multidimensional NMR data for backbone and side-chain assignments

• Cryo-EM for oligomeric complexes:

  • If CTC_00911 forms larger complexes with other operon proteins

  • May be particularly valuable if crystallization proves challenging

  • Can reveal dynamic conformational states

• HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry):

  • Map regions involved in ligand binding or protein-protein interactions

  • Compare conformational dynamics in different conditions

The choice between these methods should be guided by initial biophysical characterization results. If thermal stability measurements (DSF) show well-defined melting transitions, as observed for well-folded proteins in V. natriegens , crystallization may be more promising.

How can comparative genomics approaches reveal the function of CTC_00911?

Leverage bioinformatics and comparative genomics to gain functional insights:

• Phylogenetic profiling:

  • Identify organisms containing CTC_00911 homologs

  • Analyze co-occurrence patterns with other genes

  • Map evolutionary relationships of UPF0303 family proteins

• Structural prediction and modeling:

  • Utilize AlphaFold2 or RoseTTAFold for de novo structure prediction

  • Compare predicted structures with known fold families

  • Identify potential active sites or binding pockets

• Genomic context analysis across species:

  • Compare operonic arrangements of CTC_00911 homologs

  • Identify conserved gene neighborhoods

  • Analyze promoter regions for regulatory elements

• Domain architecture analysis:

  • Identify hidden domains or motifs

  • Compare with other uncharacterized protein families (UPFs)

  • Look for similarities to SIS domain proteins found in APIs

This approach has proven valuable for other hypothetical proteins, such as the identification of API homologs in Gram-positive bacteria that were initially found through BLASTP searches starting with the E. coli CFT037 c3406 protein .

What is the most effective experimental design to determine if CTC_00911 complements known metabolic deficiencies?

To test functional complementation, consider this comprehensive experimental design:

• Bacterial complementation assays:

  • Transform CTC_00911 expression plasmids into metabolic mutant strains

  • Similar to the approach used for CtAPI complementation in TCM15 cells (deficient in API activity)

  • Test growth restoration under selective conditions

• Heterologous expression systems:

  • Express CTC_00911 in yeast or bacterial knockout models

  • Create knockout/knockdown models of homologous genes in model organisms

  • Assess phenotypic rescue with CTC_00911

• Metabolic flux analysis:

  • Measure changes in metabolic pathways upon CTC_00911 expression

  • Use 13C-labeled substrates to track carbon flow

  • Compare flux distributions between wild-type and CTC_00911-expressing cells

• Growth condition screening:

  • Test complementation under various carbon sources

  • Vary temperature, pH, and other environmental factors

  • Identify specific conditions where CTC_00911 confers advantage

For plasmid construction, use a similar approach to that employed for CtAPI: clone CTC_00911 into vectors with leaky T7 promoters (e.g., pT7-7) to ensure appropriate expression levels for complementation studies .

What advanced protein labeling and interaction techniques would be most informative for CTC_00911?

To thoroughly investigate CTC_00911 interactions and dynamics, employ these advanced techniques:

• In vivo crosslinking and proximity labeling:

  • BioID or TurboID fusion for proximity-dependent biotinylation

  • Photo-amino acid incorporation for UV-inducible crosslinking

  • APEX2 fusion for peroxidase-based proximity labeling

• Advanced isotopic labeling strategies:

  • Site-specific incorporation of NMR-active amino acids

  • Segmental isotopic labeling for domain-specific studies

  • FRET pair incorporation for conformational studies

• In-cell NMR:

  • Express isotopically labeled CTC_00911 in live cells

  • Monitor structural changes and interactions in cellular environment

  • Compare E. coli vs. V. natriegens expression systems

• Surface plasmon resonance (SPR) and microscale thermophoresis (MST):

  • Quantify binding affinities to potential substrates

  • Measure interactions with other operon proteins

  • Determine kinetic parameters of molecular interactions

V. natriegens has shown particular advantages for isotopic labeling of challenging proteins. For 15N-labeling in V. natriegens, use ModM9 medium with 15NH4Cl as the sole nitrogen source, inducing expression at OD600 = 0.5 with 1 mM IPTG . This approach has been successfully applied to proteins that were prohibitively expensive to produce in labeled form using E. coli .

Comparative Expression System Performance for Hypothetical Proteins

Purification Strategy Comparison for UPF Family Proteins

Thermal Stability Comparison of Proteins Expressed in Different Systems

Recommended Research Workflow

• Bioinformatic analysis:

  • Sequence analysis, structural prediction, and genomic context comparison

• Recombinant expression optimization:

  • Parallel testing in E. coli and V. natriegens

  • Optimization of constructs and conditions

• Functional screening:

  • Enzymatic assays based on operon context

  • Complementation studies in suitable model systems

  • Interaction partner identification

• Structural characterization:

  • Biophysical characterization and stability assessment

  • Advanced structural determination by X-ray crystallography or NMR

This systematic approach leverages the advantages of both traditional and emerging expression systems while focusing experimental design on the most likely functional roles based on genomic context.