Recombinant Uncharacterized 80 kDa protein

What are uncharacterized proteins and why are they important in research?

Uncharacterized proteins are gene products with unknown or incompletely defined functions, structures, or biochemical properties. They represent significant research opportunities for novel discoveries in biological processes and potential therapeutic targets. Approximately 20-40% of predicted proteins in sequenced genomes remain uncharacterized, representing a vast reservoir of biological functions awaiting discovery. For example, the uncharacterized 82 kDa protein SANBR (SANT and BTB domain regulator of CSR) was recently identified as a negative regulator of Class Switch Recombination (CSR) in B cells, demonstrating how characterizing such proteins can reveal previously unknown regulatory mechanisms in immune responses .

How are recombinant uncharacterized 80 kDa proteins typically expressed and purified?

Recombinant uncharacterized 80 kDa proteins are commonly expressed in bacterial systems such as E. coli, using affinity tags to facilitate purification. The methodology typically involves:

• Gene cloning into appropriate expression vectors with affinity tags (commonly His-tag)

• Transformation into expression host cells (E. coli is common for initial studies)

• Induction of protein expression (often using IPTG or similar inducers)

• Cell lysis and protein extraction

• Affinity chromatography purification

• Further purification steps as needed (ion exchange, size exclusion)

For example, the uncharacterized 80 kDa protein from Paramecium primaurelia (product RFL35282PF) is expressed in E. coli with an N-terminal His-tag, facilitating purification through affinity chromatography . Similarly, for the 82 kDa SANBR protein, researchers expressed the recombinant BTB domain to study its characteristic properties including homodimerization and interaction with corepressor proteins .

What are optimal storage conditions for recombinant uncharacterized 80 kDa proteins?

Optimal storage conditions typically include:

• Storage temperature: -20°C to -80°C for long-term storage

• Buffer composition: Tris/PBS-based buffers with stabilizing agents

• Cryoprotectants: Addition of glycerol (5-50%) to prevent freeze-thaw damage

• Aliquoting: Division into single-use aliquots to avoid repeated freeze-thaw cycles

• Short-term storage: 4°C for up to one week for working aliquots

For the specific uncharacterized 80 kDa protein from Paramecium primaurelia, storage recommendations include:

• Long-term storage at -20°C/-80°C

• Use of Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• Reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (typically 50%) for long-term storage

• Avoiding repeated freeze-thaw cycles

What initial characterization experiments should be performed on an uncharacterized 80 kDa protein?

Initial characterization of uncharacterized proteins should follow a systematic approach:

• Biophysical characterization:

  • SDS-PAGE for purity assessment and molecular weight confirmation

  • Circular dichroism (CD) for secondary structure analysis

  • Fluorescence spectroscopy for tertiary structure insights

  • Dynamic light scattering (DLS) for homogeneity and aggregation state

• Sequence-based analysis:

  • Identification of conserved domains (like the SANT and BTB domains in SANBR)

  • Phylogenetic analysis to identify orthologs in other species

  • Prediction of post-translational modifications

  • Structural prediction using tools like Phyre2 (as used for SANBR protein)

• Basic biochemical assays:

  • Stability under various pH and temperature conditions

  • Oligomerization state (native PAGE, size exclusion chromatography)

  • Binding partners through pull-down assays

For instance, the SANBR protein was initially characterized by identifying its SANT domain (amino acids 21-59) and BTB domain (amino acids 147-255) through sequence alignment by BLAST and structure prediction by Phyre2 .

How can domain-specific functions be assessed in uncharacterized proteins?

Domain-specific functional analysis requires a targeted approach:

For example, researchers studying the SANBR protein expressed and purified its recombinant BTB domain separately to demonstrate its characteristic properties of homodimerization and interaction with corepressor proteins including HDAC and SMRT. They also performed domain deletion studies showing that the BTB domain was essential for inhibition of CSR, while the SANT domain was largely dispensable for this function .

What approaches can identify potential binding partners of uncharacterized 80 kDa proteins?

Several complementary approaches can identify binding partners:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Expression of tagged protein in relevant cell systems

  • Pull-down of protein complexes under native conditions

  • Mass spectrometric identification of co-purified proteins

  • Validation of interactions through reciprocal pull-downs

• Yeast two-hybrid (Y2H) screening:

  • Library screening to identify direct protein-protein interactions

  • Confirmation of interactions through co-immunoprecipitation

• Proximity-based labeling methods:

  • BioID or APEX2 tagging for in vivo proximity labeling

  • Identification of proteins in the same subcellular neighborhood

• In vitro binding assays:

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI)

  • Isothermal titration calorimetry (ITC) for binding kinetics and thermodynamics

For the SANBR protein, researchers identified its interactions with corepressor proteins including HDAC and SMRT, which provided insights into its mechanism of action in regulating CSR .

How can researchers determine the cellular function of an uncharacterized 80 kDa protein?

Determining cellular function requires multilevel approaches:

• Cellular localization studies:

  • Fluorescent protein tagging for live-cell imaging

  • Immunofluorescence with specific antibodies

  • Subcellular fractionation followed by western blotting

• Gene perturbation studies:

  • CRISPR/Cas9-mediated knockout or knockin

  • RNAi-mediated knockdown

  • Overexpression studies

  • Phenotypic analysis following perturbation

• High-throughput screening approaches:

  • shRNA library screens (as used for SANBR identification)

  • CRISPR screens for functional genomics

  • Chemical genetic screens

• Transcriptomic and proteomic profiling:

  • RNA-seq following gene perturbation

  • Proteomics to identify changes in protein networks

  • Phosphoproteomics for signaling pathway impacts

For example, SANBR was identified as a negative regulator of Class Switch Recombination using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line. Further functional validation included overexpression studies in primary mouse splenic B cells, which confirmed SANBR's inhibitory effect on CSR .

What strategies can resolve conflicting data in uncharacterized protein research?

Resolving conflicting data requires systematic troubleshooting:

• Methodological validation:

  • Cross-validation using orthogonal techniques

  • Careful examination of experimental conditions

  • Reproduction of experiments with standardized protocols

• Protein context considerations:

  • Cell type-specific effects and expression patterns

  • Post-translational modifications affecting function

  • Binding partners present in different experimental systems

• Structural considerations:

  • Protein conformation differences in varying conditions

  • Tags potentially affecting protein function

  • Domain interactions and protein dynamics

• Integrated data analysis:

  • Meta-analysis of available data

  • Statistical reanalysis of quantitative data

  • Consideration of biological variability

When researchers encounter conflicting results about protein function, they should systematically evaluate experimental conditions, cell-specific contexts, and potential technical artifacts. For example, if protein function differs between in vitro and cellular studies, considerations about proper folding, missing cofactors, or post-translational modifications may resolve these discrepancies.

How can structural analysis guide functional studies of uncharacterized 80 kDa proteins?

Structural analysis provides crucial insights for functional studies:

• Structure prediction and modeling:

  • Homology modeling based on related proteins

  • Ab initio modeling for novel folds

  • Integration of experimental data with computational predictions

• Low-resolution structural analysis:

  • Small-angle X-ray scattering (SAXS)

  • Negative stain electron microscopy

  • Chemical crosslinking coupled with mass spectrometry

• High-resolution structure determination:

  • X-ray crystallography

  • Cryo-electron microscopy

  • NMR spectroscopy for smaller domains

• Structure-guided functional studies:

  • Rational design of mutations based on structural insights

  • Identification of potential binding sites or catalytic residues

  • Design of domain-specific functional assays

For the SANBR protein, structure prediction using Phyre2 revealed its SANT and BTB domains, guiding subsequent functional studies that demonstrated the BTB domain's importance in protein-protein interactions and CSR inhibition .

What are the optimal expression systems for difficult-to-express 80 kDa recombinant proteins?

Expression system selection depends on protein properties:

• Prokaryotic systems:

  • Standard E. coli strains (BL21, Rosetta) for initial attempts

  • Specialized strains for disulfide bond formation (Origami, SHuffle)

  • Cold-inducible systems for improved folding (Arctic Express)

• Eukaryotic systems:

  • Yeast (Pichia pastoris, S. cerevisiae) for proteins requiring post-translational modifications

  • Insect cells (Sf9, High Five) for complex mammalian proteins

  • Mammalian cells (HEK293, CHO) for highest authenticity of mammalian proteins

• Cell-free systems:

  • Wheat germ extracts for difficult-to-express proteins

  • E. coli-based cell-free systems for rapid screening

• Expression optimization strategies:

  • Codon optimization

  • Fusion tags (SUMO, MBP, GST) to enhance solubility

  • Expression as protein fragments

  • Chaperone co-expression

For example, the uncharacterized 80 kDa protein from Paramecium primaurelia was successfully expressed in E. coli with a His-tag , while for proteins requiring more complex folding or post-translational modifications, eukaryotic expression systems might be more appropriate.

How can researchers develop specific antibodies against uncharacterized 80 kDa proteins?

Antibody development strategies include:

• Antigen preparation:

  • Full-length recombinant protein if soluble

  • Soluble domains for large proteins

  • Synthetic peptides for specific regions

  • Consideration of native structure and accessibility

• Immunization strategies:

  • Multiple host animals for diverse antibody repertoire

  • Adjuvant selection to enhance immunogenicity

  • Prime-boost protocols for high-affinity antibodies

• Antibody screening and validation:

  • ELISA for initial screening

  • Western blotting against recombinant and native protein

  • Immunoprecipitation to verify native protein recognition

  • Immunofluorescence for subcellular localization studies

  • Validation in knockout/knockdown models

• Monoclonal antibody development:

  • Hybridoma technology or phage display

  • Single B-cell cloning approaches

  • Humanization for therapeutic applications

For validation of protein expression in recombinant systems, researchers studying the SANBR protein used western blot analysis with anti-Flag, anti-CD80, or anti-CTB antibodies to detect the expression of their recombinant proteins .

What protocols are most effective for functional reconstitution of uncharacterized 80 kDa proteins?

Functional reconstitution requires careful consideration of protein environment:

• Buffer optimization:

  • Systematic screening of buffer components (pH, salt, additives)

  • Inclusion of stabilizing agents based on protein characteristics

  • Mimicking physiological conditions when possible

• Cofactor identification and incorporation:

  • Bioinformatic prediction of potential cofactors

  • Testing metal ions, nucleotides, or other small molecules

  • Reconstitution with predicted cofactors

• Interaction partners:

  • Co-expression with binding partners

  • Reconstitution with purified interaction partners

  • Assembly of multiprotein complexes in vitro

• Membrane protein considerations:

  • Detergent screening for extraction and purification

  • Reconstitution into liposomes or nanodiscs

  • Use of amphipols or other membrane mimetics

For example, the recombinant BTB domain of SANBR was functionally reconstituted to demonstrate its characteristic homodimerization and interaction with corepressor proteins including HDAC and SMRT .

How can mass spectrometry be utilized to characterize uncharacterized 80 kDa proteins?

Mass spectrometry offers powerful characterization capabilities:

• Protein identification and verification:

  • Peptide mass fingerprinting

  • Sequence coverage analysis

  • Post-translational modification mapping

• Structural characterization:

  • Hydrogen-deuterium exchange (HDX-MS) for conformational dynamics

  • Chemical crosslinking MS for proximity mapping

  • Native MS for intact complex analysis and stoichiometry determination

• Protein-protein interactions:

  • Affinity purification-MS (AP-MS)

  • Proximity labeling coupled with MS

  • Protein correlation profiling

• Functional analyses:

  • Activity-based protein profiling

  • Thermal proteome profiling for ligand binding

  • Cellular thermal shift assay coupled with MS (MS-CETSA)

Advanced mass spectrometry techniques have revolutionized the study of uncharacterized proteins. As noted in search result , mass-tolerant database searching can identify a large proportion of previously unassigned spectra in shotgun proteomics as modified peptides, enhancing characterization capabilities .

What bioinformatic approaches best predict functions of uncharacterized 80 kDa proteins?

Bioinformatic prediction employs multiple complementary strategies:

• Sequence-based prediction:

  • Homology searching (BLAST, HMMER)

  • Conserved domain identification (Pfam, InterPro)

  • Motif analysis for functional sites

  • Remote homology detection (HHpred, FFAS)

• Structure-based prediction:

  • Homology modeling (SWISS-MODEL, Phyre2)

  • Threading approaches (I-TASSER)

  • Ab initio modeling (Rosetta)

  • Active site prediction based on structural features

• Network-based prediction:

  • Guilt-by-association approaches

  • Co-expression network analysis

  • Protein-protein interaction predictions

  • Phylogenetic profiling

• Integrated approaches:

  • Machine learning methods combining multiple features

  • Confidence scoring of predictions

  • Experimental validation of top predictions

For the uncharacterized SANBR protein, researchers used BLAST for sequence alignment and Phyre2 for structure prediction to identify its SANT domain (amino acids 21-59) and BTB domain (amino acids 147-255), which guided subsequent functional studies .

How can researchers differentiate between direct and indirect effects in functional studies of uncharacterized proteins?

Differentiating direct and indirect effects requires controlled experimental designs:

• In vitro reconstitution:

  • Purified component systems to demonstrate direct effects

  • Stepwise addition of components to identify minimal requirements

  • Kinetic analyses to establish order of events

• Targeted mutagenesis:

  • Structure-guided mutations of predicted functional residues

  • Separation-of-function mutations

  • Rescue experiments with mutant proteins

• Temporal resolution studies:

  • Rapid induction or inhibition systems (e.g., auxin-inducible degron)

  • Time-course experiments to establish causality

  • Pulse-chase approaches for dynamic processes

• Proximity-based methods:

  • FRET/BRET for direct interactions in live cells

  • Proximity ligation assays for endogenous proteins

  • Split-protein complementation assays

For example, researchers studying SANBR performed domain deletion studies and demonstrated that inhibition of CSR is dependent specifically on the BTB domain while the SANT domain is largely dispensable, helping to establish a direct mechanistic link .

How can functional characterization of uncharacterized 80 kDa proteins advance disease understanding?

Functional characterization can impact disease research through:

• Disease mechanism elucidation:

  • Identification of proteins involved in pathological pathways

  • Characterization of disease-associated variants

  • Understanding of pathway dysregulation in disease states

• Biomarker development:

  • Validation of uncharacterized proteins as disease indicators

  • Development of detection methods for clinical application

  • Correlation of protein levels with disease progression

• Therapeutic target identification:

  • Validation of druggability

  • Development of screening assays

  • Identification of interaction surfaces for drug design

• Pathway analysis:

  • Integration of newly characterized proteins into pathway models

  • Systems biology approaches to understand network effects

  • Identification of novel regulatory mechanisms

The discovery of SANBR as a negative regulator of Class Switch Recombination provides insights into immune regulation that could be relevant for understanding immune disorders, as proper resolution of CSR prevents damage due to uncontrolled and prolonged immune responses .

What strategies can translate basic research on uncharacterized proteins into therapeutic applications?

Translational research strategies include:

• Target validation approaches:

  • Animal models with genetic modifications

  • Disease-relevant cellular systems

  • Human genetics correlations

• Development of modulators:

  • High-throughput screening for small molecule inhibitors/activators

  • Fragment-based drug design

  • Structure-based rational design

  • Biologics development (antibodies, peptides)

• Delivery system development:

  • Targeting specific tissues or cell types

  • Overcoming cellular barriers

  • Improving stability and pharmacokinetics

• Preclinical testing:

  • Efficacy in disease models

  • Toxicity assessment

  • Pharmacokinetic/pharmacodynamic studies

For example, the study of hsCD80 expressed by recombinant Lactococcus lactis demonstrated promising antitumor effects by priming active antitumor immunity and restoring T cell activity in colorectal cancer models . This illustrates how characterization of previously uncharacterized proteins can lead to novel therapeutic approaches.

How can researchers improve solubility and stability of recombinant uncharacterized 80 kDa proteins?

Solubility and stability enhancement strategies include:

• Expression optimization:

  • Lower induction temperature (16-25°C)

  • Reduced inducer concentration

  • Co-expression with chaperones

  • Solubility-enhancing fusion tags (SUMO, MBP, GST)

• Buffer optimization:

  • Systematic pH screening

  • Salt type and concentration variations

  • Addition of stabilizing agents (glycerol, arginine, trehalose)

  • Reducing agents for proteins with cysteines

• Protein engineering approaches:

  • Surface entropy reduction

  • Removal of aggregation-prone regions

  • Disulfide bond engineering

  • Domain-based expression

• Storage condition optimization:

  • Flash-freezing techniques

  • Lyophilization with appropriate excipients

  • Addition of cryoprotectants

For the uncharacterized 80 kDa protein from Paramecium primaurelia, researchers optimized storage in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 and recommended addition of 5-50% glycerol for long-term storage .

What are effective strategies for resolving expression and purification challenges with uncharacterized proteins?

Expression and purification troubleshooting involves:

• Expression troubleshooting:

  • Codon optimization for expression host

  • Alternative vector systems

  • Testing multiple expression hosts

  • Induction parameter optimization

  • Expression as protein fragments

• Solubility enhancement:

  • Detergent screening for membrane or hydrophobic proteins

  • Denaturation and refolding approaches

  • Co-expression with binding partners

  • Cell-free expression systems

• Purification optimization:

  • Multiple orthogonal purification steps

  • On-column refolding

  • Size exclusion chromatography for final polishing

  • Removal of aggregates and degradation products

• Quality control measures:

  • Dynamic light scattering for homogeneity

  • Thermal shift assays for stability assessment

  • Activity assays for functional verification

  • Mass spectrometry for identity confirmation

For example, in the study of recombinant hsCD80 or CTB-hsCD80 expressed in L. lactis, researchers analyzed different concentrations of the inducer (nisin) at different induction times to optimize expression conditions, finding that 2 ng/ml nisin for 6 hours provided maximum expression levels .

How can researchers develop reliable functional assays for proteins with unknown functions?

Developing functional assays for uncharacterized proteins involves:

• Hypothesis-driven approaches:

  • Domain prediction to suggest potential functions

  • Structural similarity to characterized proteins

  • Subcellular localization to inform potential roles

  • Protein interaction partners to suggest pathway involvement

• Unbiased screening approaches:

  • Phenotypic screens following protein perturbation

  • Cellular response profiling

  • Metabolic profiling

  • Interactome analysis

• Biochemical activity testing:

  • Enzymatic activity screens

  • Binding assays with potential substrates or partners

  • Structural changes upon ligand addition

  • Thermal shift assays to identify stabilizing ligands

• Computational prediction validation:

  • Testing predicted substrates or interactions

  • Structure-based function prediction validation

  • Network-based function prediction testing

For the SANBR protein, researchers developed functional assays based on its predicted role in CSR, including in vitro studies of its purified BTB domain to demonstrate homodimerization and interaction with corepressor proteins, as well as in vivo studies showing that overexpression inhibited CSR in primary mouse splenic B cells .

What emerging technologies will advance the study of uncharacterized proteins?

Emerging technologies with significant potential include:

• Advanced structural methods:

  • Cryo-electron microscopy for challenging proteins

  • Integrative structural biology approaches

  • AlphaFold and other AI-based structure prediction

  • Single-molecule techniques for conformational dynamics

• High-throughput functional screening:

  • CRISPR/Cas9-based genetic screens

  • Pooled protein expression libraries

  • Automated phenotypic screening platforms

  • Deep mutational scanning

• Single-cell technologies:

  • Single-cell proteomics

  • Spatial transcriptomics and proteomics

  • Live-cell imaging with advanced biosensors

  • Single-molecule tracking in live cells

• Computational biology advances:

  • Machine learning for function prediction

  • Network-based analyses

  • Systems biology integration

  • Molecular dynamics simulations at extended timescales

Technologies such as mass-tolerant database searching have already shown promise in identifying a large proportion of previously unassigned spectra in shotgun proteomics as modified peptides, enhancing our ability to characterize proteins .

How can multi-omics approaches enhance understanding of uncharacterized protein functions?

Multi-omics integration offers comprehensive insights:

• Data integration strategies:

  • Correlation analyses across omics datasets

  • Network inference from multi-omics data

  • Machine learning approaches for pattern recognition

  • Causal network modeling

• Complementary omics applications:

  • Genomics for genetic context and variation

  • Transcriptomics for expression patterns and regulation

  • Proteomics for abundance, PTMs, and interactions

  • Metabolomics for functional endpoints

• Temporal and spatial considerations:

  • Developmental time course analyses

  • Tissue- and cell-specific profiling

  • Subcellular compartment analysis

  • Response to perturbations across omics layers

• Functional validation of multi-omics predictions:

  • Targeted genetic manipulations

  • Biochemical validation of predicted activities

  • Cellular phenotype confirmation

For example, researchers studying the SANBR protein combined genetic screening (shRNA library) with biochemical characterization and cellular functional studies to establish its role as a negative regulator of CSR .

What are the ethical considerations in research on uncharacterized proteins with potential therapeutic applications?

Ethical considerations include:

• Research integrity aspects:

  • Transparent reporting of negative and positive results

  • Careful validation before function assignment

  • Reproducibility considerations

  • Data and material sharing

• Translational research ethics:

  • Appropriate preclinical testing before clinical applications

  • Consideration of off-target effects

  • Risk-benefit assessment for novel therapeutics

  • Informed consent for clinical trials

• Intellectual property considerations:

  • Balancing protection with knowledge advancement

  • Collaborative research agreements

  • Technology transfer to enhance accessibility

  • Open science initiatives

• Broader societal impacts:

  • Equitable access to resulting therapeutics

  • Consideration of global health needs

  • Environmental impacts of production methods

  • Potential dual-use concerns