Recombinant Mouse Periodic tryptophan protein 2 homolog (Pwp2), partial

What is the structure and function of mouse Pwp2?

Mouse Periodic tryptophan protein 2 homolog (Pwp2) is a WD40 repeat-containing protein that functions as a core component of the small subunit (SSU) processome. The protein plays a critical role in ribosome biogenesis through:

• Facilitating pre-18S rRNA processing

• Mediating interactions within the U3 snoRNP complex

• Serving as a scaffold protein that recruits other processing factors

The WD40 repeat domains form a β-propeller structure that creates a stable platform for protein-protein interactions, which is essential for its function in the multiprotein complexes involved in ribosomal assembly.

When working with recombinant mouse Pwp2, researchers should consider both the full-length protein (typically around 100-105 kDa) and partial constructs that contain the functional domains of interest.

What expression systems are most suitable for recombinant mouse Pwp2 production?

Several expression systems can be used for producing recombinant mouse Pwp2, each with specific advantages:

For structural studies requiring large quantities of protein, bacterial expression systems like E. coli are often preferred, similar to production methods used for other recombinant mouse proteins . For functional studies where post-translational modifications are critical, mammalian or insect cell systems may be more appropriate.

How should recombinant mouse Pwp2 be stored to maintain stability?

Optimal storage conditions for recombinant mouse Pwp2 include:

• Long-term storage at -80°C in small single-use aliquots

• Addition of stabilizing agents such as 10-15% glycerol

• Inclusion of reducing agents (e.g., DTT or β-mercaptoethanol) at 1-5 mM to prevent oxidation of cysteine residues

• Storage buffer at pH 7.5-8.0 (typically Tris or phosphate-based)

Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity. When working with the protein, keep it on ice and use within 24 hours of thawing for optimal results.

What are the optimal conditions for studying Pwp2's role in pre-ribosomal complexes?

To effectively study Pwp2's function in pre-ribosomal complexes:

• Co-immunoprecipitation (Co-IP) assays:

  • Use mild lysis buffers containing 20-50 mM HEPES pH 7.4, 100-150 mM NaCl, 0.1-0.5% NP-40

  • Include protease inhibitors and phosphatase inhibitors to preserve complex integrity

  • Perform at 4°C to maintain native interactions

• Sucrose gradient centrifugation:

  • Use 10-50% sucrose gradients in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂

  • Centrifuge at 170,000-200,000 × g for 2.5-3 hours

  • Collect fractions and analyze by Western blotting to track Pwp2 association with ribosomal precursors

• Proximity labeling approaches:

  • Use BioID or TurboID fusions with Pwp2 to identify transient interactions

  • Express in relevant cell lines for 16-24 hours before biotin addition

  • Perform streptavidin pulldown followed by mass spectrometry analysis

These approaches require careful optimization, similar to those used for other nuclear proteins involved in multiprotein complexes .

How can researchers analyze Pwp2 knockout or knockdown effects on ribosome biogenesis?

When studying the effects of Pwp2 depletion on ribosome biogenesis:

• Northern blot analysis:

  • Extract total RNA using TRIzol or similar reagents

  • Use specific probes for pre-rRNA intermediates (internal transcribed spacers)

  • Quantify accumulation of precursors to identify processing defects

• Polysome profiling:

  • Prepare cell lysates in polysome buffer (20 mM HEPES pH 7.6, 100 mM KCl, 5 mM MgCl₂, 100 μg/ml cycloheximide)

  • Fractionate on 10-50% sucrose gradients

  • Monitor A254 during fractionation to generate polysome profiles

  • Compare profiles between control and Pwp2-depleted cells

• Fluorescence microscopy:

  • Use FISH (fluorescence in situ hybridization) with rRNA-specific probes

  • Alternatively, use immunofluorescence to track nucleolar markers

  • Quantify nucleolar disruption or pre-rRNA mislocalization

These approaches provide complementary information about the impact of Pwp2 depletion on various aspects of ribosome biogenesis.

What techniques can be used to characterize post-translational modifications of Pwp2?

Characterizing post-translational modifications (PTMs) of Pwp2 requires specialized approaches:

• Mass spectrometry-based analysis:

  • Perform immunoprecipitation of Pwp2 from cell lysates

  • Digest with trypsin or other proteases

  • Analyze by LC-MS/MS with PTM-specific search parameters

  • Consider enrichment strategies for phosphopeptides (TiO₂, IMAC) or ubiquitinated peptides

• Site-directed mutagenesis:

  • Identify potential modification sites by sequence analysis or mass spectrometry

  • Generate point mutations (e.g., S→A for phosphorylation sites)

  • Assess functional consequences through complementation assays

• Western blotting with modification-specific antibodies:

  • Use phospho-specific, acetylation-specific, or ubiquitin-specific antibodies

  • Compare modification patterns across different cell types or conditions

  • Confirm specificity using phosphatase treatment or deacetylase treatment as controls

PTM analysis should incorporate appropriate controls, including enzyme inhibitors during sample preparation to preserve labile modifications.

How can researchers validate the purity and integrity of recombinant mouse Pwp2?

Multiple complementary techniques should be used to assess protein quality:

• SDS-PAGE analysis:

  • Run protein samples on 8-10% gels (given Pwp2's size)

  • Stain with Coomassie Blue or silver stain

  • Look for a single band at the expected molecular weight (~100-105 kDa for full-length)

  • Assess purity by densitometry (aim for >90% purity)

• Western blotting:

  • Use anti-Pwp2 antibodies or anti-tag antibodies if the recombinant protein contains tags

  • Confirm the absence of degradation products

  • Verify correct molecular weight

• Mass spectrometry:

  • Perform peptide mass fingerprinting

  • Confirm sequence coverage (aim for >80%)

  • Check for unexpected modifications or truncations

• Dynamic light scattering:

  • Assess sample homogeneity and aggregation state

  • Determine hydrodynamic radius

  • Identify potential oligomerization

Similar quality control approaches are standard practice for recombinant proteins as seen with other mouse proteins described in the literature .

What functional assays can verify the activity of recombinant mouse Pwp2?

Validating the functional activity of recombinant Pwp2 is crucial before using it in downstream applications:

• RNA binding assays:

  • Electrophoretic mobility shift assay (EMSA) with pre-rRNA segments

  • Filter binding assays

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Protein interaction verification:

  • Pull-down assays with known binding partners

  • Size exclusion chromatography to analyze complex formation

  • Isothermal titration calorimetry (ITC) to determine binding constants

• Complementation assays:

  • Express recombinant Pwp2 in Pwp2-depleted cells

  • Assess rescue of ribosome biogenesis defects

  • Measure restoration of cell growth

These assays provide different but complementary information about the functionality of the recombinant protein.

How should researchers design constructs for domain analysis of mouse Pwp2?

When designing constructs for domain-specific analysis:

• In silico domain prediction:

  • Use tools like SMART, Pfam, or InterPro to identify conserved domains

  • Analyze secondary structure predictions to avoid disrupting structural elements

  • Consider disordered regions that may be important for function

• Construct design principles:

  • Include complete domains rather than partial domains

  • Add short linkers (3-5 amino acids) between the tag and protein

  • Consider solubility-enhancing tags for difficult domains

  • Generate both N- and C-terminally tagged versions to determine optimal configuration

• Validation approaches:

  • Test expression levels of multiple constructs in parallel

  • Assess solubility using small-scale purification

  • Verify folding using circular dichroism or limited proteolysis

A systematic approach to construct design significantly increases the likelihood of obtaining functional protein domains for structural and interaction studies.

What are the critical considerations when designing Pwp2 knockout or knockdown experiments?

When depleting Pwp2 in experimental systems:

• CRISPR/Cas9 knockout strategy:

  • Design guide RNAs targeting early exons

  • Consider conditional knockout systems due to potential essentiality

  • Verify knockout by sequencing and Western blotting

  • Establish rescue lines expressing recombinant Pwp2 to confirm specificity

• siRNA/shRNA knockdown approach:

  • Test multiple siRNA sequences for efficacy and specificity

  • Optimize transfection conditions for target cell type

  • Include non-targeting control siRNAs

  • Validate knockdown efficiency by qRT-PCR and Western blotting

• Timing considerations:

  • Monitor effects at multiple time points (24, 48, 72 hours)

  • Consider cell cycle effects, as ribosome biogenesis is cell cycle-regulated

  • Be aware of potential compensatory mechanisms in long-term depletion studies

• Phenotypic analysis:

  • Assess cell growth and proliferation

  • Analyze nucleolar morphology

  • Evaluate rRNA processing through Northern blotting

  • Examine global protein synthesis using metabolic labeling

Careful experimental design with appropriate controls is essential to distinguish direct effects of Pwp2 depletion from secondary consequences.

How can researchers investigate the structural characteristics of recombinant mouse Pwp2?

Several complementary approaches can provide structural insights:

Integrating data from multiple structural techniques provides the most comprehensive understanding of Pwp2's structure-function relationship.

What approaches are effective for studying Pwp2 interactions within the pre-ribosomal complex?

To study Pwp2's interactions within complex assemblies:

• Crosslinking mass spectrometry (XL-MS):

  • Use crosslinkers of different lengths (e.g., DSS, BS3, EDC)

  • Apply to intact cells or purified complexes

  • Analyze crosslinked peptides by specialized mass spectrometry methods

  • Provides direct evidence of spatial proximity within complexes

• Cryo-electron tomography:

  • Visualize pre-ribosomal complexes in their cellular context

  • Use gold-labeled antibodies against Pwp2 for localization

  • Perform subtomogram averaging for higher resolution

• ChIP-seq and CLIP-seq:

  • ChIP-seq to identify chromatin association at rDNA loci

  • CLIP-seq to map RNA binding sites with nucleotide resolution

  • Require optimization of crosslinking and immunoprecipitation conditions

• Proximity labeling in living cells:

  • Express Pwp2 fused to BioID or TurboID

  • Allow biotinylation of nearby proteins in vivo

  • Purify biotinylated proteins and identify by mass spectrometry

  • Provides temporal information about dynamic interactions

These techniques provide complementary information about Pwp2's position and interactions within the pre-ribosomal complex, helping to establish its precise role in ribosome assembly.

How can researchers address low expression yields of recombinant mouse Pwp2?

When facing challenges with protein expression:

• Optimize codon usage:

  • Adapt codons to the expression host

  • Remove rare codons, especially at the N-terminus

  • Consider GC content and mRNA secondary structure

• Adjust expression conditions:

  • Test multiple induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Vary inducer concentration (IPTG: 0.1-1.0 mM for bacterial systems)

  • Try different media formulations (TB, 2XYT, auto-induction media)

  • Optimize expression duration (3 hours to overnight)

• Consider fusion partners:

  • Test solubility-enhancing partners (MBP, SUMO, GST, TrxA)

  • Include precision protease sites for tag removal

  • Compare N-terminal vs. C-terminal tag placement

• Expression host selection:

  • Try specialized E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Consider alternative expression systems if bacterial expression fails

  • Test insect cell lines (Sf9, High Five) for baculovirus expression

A systematic approach to optimization, testing multiple variables in parallel, can significantly improve recombinant protein yields.

What strategies can resolve aggregation issues with purified Pwp2?

To address protein aggregation problems:

• Buffer optimization:

  • Screen different pH values (typically 6.5-8.5)

  • Test various salt concentrations (100-500 mM NaCl)

  • Include stabilizing additives (glycerol, arginine, trehalose)

  • Add reducing agents (DTT, TCEP) to prevent disulfide-mediated aggregation

• Purification strategy adjustments:

  • Include detergents during lysis (0.1% Triton X-100 or NP-40)

  • Perform purification at 4°C throughout

  • Consider on-column refolding for inclusion body purification

  • Use size exclusion chromatography as a final step to remove aggregates

• Protein engineering approaches:

  • Remove hydrophobic patches identified by in silico analysis

  • Introduce solubilizing mutations based on homology models

  • Express isolated domains rather than full-length protein

  • Use fusion partners known to enhance solubility

• Analytical techniques to monitor aggregation:

  • Dynamic light scattering to detect early aggregation

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Differential scanning fluorimetry to assess thermal stability

Systematic optimization is key to resolving aggregation issues, with parallel testing of multiple conditions recommended.

How can researchers employ recombinant Pwp2 for drug discovery applications?

Using recombinant Pwp2 in drug discovery efforts:

• High-throughput screening approaches:

  • Develop fluorescence polarization assays for RNA or protein binding

  • Establish FRET-based interaction assays for complex formation

  • Use differential scanning fluorimetry to identify stabilizing compounds

  • Implement surface plasmon resonance for direct binding studies

• Structure-based drug design:

  • Use solved structures to identify potential binding pockets

  • Perform in silico docking studies to identify candidate compounds

  • Validate hits with biophysical assays (ITC, SPR, MST)

  • Optimize lead compounds through medicinal chemistry

• Cellular assay development:

  • Create reporter systems for Pwp2 function in cells

  • Develop high-content imaging assays for nucleolar disruption

  • Establish growth inhibition assays in Pwp2-dependent cell lines

  • Generate engineered cell lines with mutations in Pwp2 binding sites

• Target validation strategies:

  • Use CRISPR/Cas9 to generate specific mutations in binding sites

  • Develop resistance models through directed evolution

  • Perform structure-activity relationship studies with compound series

  • Use chemical genetics approaches with engineered alleles

These approaches can help identify compounds that modulate Pwp2 function, potentially providing tools for studying ribosome biogenesis or developing therapeutics targeting this process.

What are the best approaches for studying Pwp2 in tissue-specific contexts?

To investigate tissue-specific functions of Pwp2:

• Conditional knockout mouse models:

  • Generate floxed Pwp2 alleles for tissue-specific deletion

  • Use appropriate Cre driver lines for targeting specific tissues

  • Analyze phenotypes at multiple developmental timepoints

  • Perform rescue experiments with wild-type or mutant Pwp2

• Primary cell culture systems:

  • Isolate primary cells from relevant tissues (e.g., liver, brain)

  • Manipulate Pwp2 expression using viral vectors or siRNA

  • Compare effects across different cell types

  • Assess tissue-specific interaction partners by co-immunoprecipitation

• Tissue-specific proteomics:

  • Perform Pwp2 immunoprecipitation from different tissue extracts

  • Identify tissue-specific interaction partners by mass spectrometry

  • Compare post-translational modification patterns across tissues

  • Use SILAC or TMT labeling for quantitative comparisons

• Single-cell approaches:

  • Apply single-cell RNA-seq to study cell-type-specific effects of Pwp2 perturbation

  • Use single-cell proteomics to examine protein-level changes

  • Perform spatial transcriptomics to map effects in tissue context

  • Employ advanced imaging to visualize Pwp2 localization in tissue sections

These approaches help determine whether Pwp2 has tissue-specific functions beyond its core role in ribosome biogenesis, potentially revealing novel regulatory mechanisms.