Recombinant Rat Protein CutA (Cuta)

What expression systems are most suitable for recombinant rat CutA protein?

Several expression systems have demonstrated efficacy for recombinant rat proteins, with selection depending on your specific experimental requirements:

For bacterial expression, E. coli-based systems using inducible promoters like the T7 RNA polymerase system have proven successful for rat proteins. This approach allows for active and selective synthesis of recombinant proteins, as demonstrated with rat TGF-β1 . The advantage is high yield, though proper folding of mammalian proteins can be challenging.

For eukaryotic expression, HEK293 cell lines offer significant advantages for rat proteins requiring post-translational modifications. This non-viral, transient transfection method is easily scalable, fast, and allows for modular construct design . The system has been successfully used to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains from rats .

For potentially higher yields, Pichia pastoris has proven effective for rat liver monoamine oxidase B expression, offering proper folding capabilities for complex rat proteins .

The optimal choice depends on CutA's specific requirements for folding, post-translational modifications, and your downstream applications.

What are the typical yields for recombinant rat proteins in different expression systems?

Yield varies considerably based on expression system and protein characteristics:

In HEK293 transient transfection systems, yields for rat C-type lectin-like receptors range from 0.2 to 5 mg per liter of production medium, with Clr-11 and RCTL representing the highest and lowest yields, respectively .

For bacterial expression, purification from inclusion bodies often occurs with significant loss, as seen in the purification of rat TGF-β1, where sequential detergent washes were required to achieve homogeneity .

How do I verify the identity and purity of recombinant rat CutA?

Multiple complementary methods should be employed:

• SDS-PAGE with Coomassie blue staining provides initial assessment of purity and molecular weight (expected to show a single band at the predicted molecular weight of CutA) .

• Western blot analysis using specific anti-CutA antibodies confirms identity. If specific antibodies are unavailable, epitope tagging (e.g., His-tag) enables detection using commercial antibodies .

• N-terminal sequencing confirms protein identity by matching amino acid sequence to the expected N-terminus of CutA, as demonstrated with recombinant rat TGF-β1 .

• Mass spectrometry provides precise molecular weight determination and can verify post-translational modifications.

• For activity verification, develop a functional assay specific to CutA's biochemical properties.

What strategies can optimize production of properly folded rat CutA for structural studies?

Proper folding is critical for structural studies, and several approaches have proven successful:

• Construct optimization: Create multiple constructs with varying N- and C-terminal boundaries to identify stable domains. For rat NK cell receptors, truncated constructs removing flexible regions improved expression and stability .

• Fusion partners: Consider fusion to Fc fragments or other solubility-enhancing domains. Fc fusion promotes proper disulfide formation in rat NK cell receptors .

• Expression conditions: Modulate temperature, induction time, and inducer concentration. For rat TGF-β1, controlled induction parameters significantly affected proper folding .

• Codon optimization: Adapt the codon usage to the expression host for improved translation efficiency.

• Glycosylation engineering: If CutA is naturally glycosylated, expression in mammalian systems with glycosylation control may be necessary, as successful N-linked glycosylation control has been demonstrated in HEK293 cells .

• Selenomethionine incorporation: For crystallography purposes, selenomethionine incorporation in HEK293 cells has been successful for rat proteins .

How can I assess the oligomeric state of recombinant rat CutA?

The oligomeric state assessment is crucial for functional and structural studies:

• Size exclusion chromatography (SEC) separates proteins based on size and shape. Compare elution volumes with molecular weight standards to determine if CutA exists as monomer, dimer, or higher-order oligomers .

• Fluorescence-detected size exclusion chromatography (FSEC) provides enhanced sensitivity for detecting oligomeric states and is particularly useful for membrane proteins and proteins expressed at low levels .

• Native PAGE enables visualization of protein complexes under non-denaturing conditions, preserving quaternary structure.

• Analytical ultracentrifugation provides precise determination of molecular weight and shape in solution.

• Dynamic light scattering (DLS) measures the hydrodynamic radius of particles in solution, useful for detecting aggregation.

• Cross-linking experiments using chemical cross-linkers can capture transient protein-protein interactions.

For rat NK cell receptors, both monomeric and dimeric forms were observed depending on the specific construct design, with dimeric forms often stabilized by disulfide bridges .

How do post-translational modifications affect recombinant rat protein function?

Post-translational modifications (PTMs) significantly impact protein function:

For rat protein C, gamma-carboxyglutamic acid residues are essential for function. Amino acid analysis confirmed the presence of 10 moles of gamma-carboxyglutamic acid residues per mol of protein . This highlights the importance of using expression systems capable of performing the necessary PTMs.

The functional impact of PTMs should be evaluated through comparative studies between recombinant and native proteins. For rat protein C, both recombinant and plasma-derived forms showed similar activation by thrombin-thrombomodulin complex, indicating proper functional PTMs in the recombinant form .

For CutA specifically, determine which PTMs are present in the native protein and evaluate whether your expression system can reproduce these modifications. Different expression systems have varying capabilities for PTMs:

• Bacterial systems: Limited PTM capability

• Yeast: Capable of many but not all mammalian PTMs

• Mammalian cells: Most complete PTM profile

What are the critical steps in optimizing crystallization of recombinant rat proteins?

Crystallization remains challenging and requires systematic optimization:

• Protein quality: Ensure monodisperse protein preparations through SEC. Heterogeneity in protein samples is a major impediment to crystallization.

• Construct optimization: Design multiple constructs removing flexible regions. For rat NK cell receptors, systematic truncation of N- and C-termini identified stable constructs amenable to crystallization .

• Surface engineering: Consider surface entropy reduction by mutating clusters of flexible, charged residues to alanines.

• Deglycosylation: If glycosylated, controlled deglycosylation or expression of non-glycosylated mutants may improve crystallization.

• Ligand co-crystallization: Including ligands or binding partners can stabilize flexible regions and promote crystal formation.

• Crystallization screening: Employ high-throughput screening with diverse precipitants, buffers, and additives at different temperatures.

• Optimization: Fine-tune promising crystallization conditions by varying protein concentration, precipitant concentration, pH, and temperature.

• Cryoprotection: Develop appropriate cryoprotection protocols to minimize damage during freezing.

For membrane proteins, addition of lipids or detergents specific to the target protein has proven beneficial .

What approaches are most effective for studying structure-function relationships in rat CutA?

Multiple complementary approaches provide comprehensive insights:

• Mutagenesis: Create point mutations or deletions to identify critical residues or domains. Systematic alanine scanning can reveal functional hotspots.

• Chimeric proteins: Exchange domains between related proteins to determine domain-specific functions, as demonstrated with rat TGF-β1 .

• Comparative analysis: Compare recombinant and native forms to ensure functional equivalence. For rat protein C, comparative studies revealed that recombinant and plasma rat PC prolonged the activated partial thromboplastin time similarly, while human PC was less effective in rat plasma .

• Structure determination: X-ray crystallography or cryo-EM provides atomic-resolution structural information. NMR can be used for smaller proteins or domains.

• Spectroscopic techniques: Circular dichroism (CD), fluorescence spectroscopy, and FTIR provide information about secondary structure and conformational changes upon ligand binding.

• Computational approaches: Molecular dynamics simulations can predict the effects of mutations or ligand binding on protein structure and dynamics.

How can I develop reliable functional assays for recombinant rat CutA?

Developing robust functional assays requires understanding CutA's biological role:

• Enzymatic activity: If CutA possesses enzymatic activity, develop assays measuring substrate conversion or product formation. For rat monoamine oxidase B, specific activity was measured in units (1 U = 1 μmol product/min) under standardized conditions .

• Binding assays: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can quantify binding affinities to interaction partners.

• Cell-based assays: Develop assays measuring cellular responses to CutA, such as signaling pathway activation or phenotypic changes.

• Comparative benchmarking: Compare your recombinant CutA with commercially available standards or native protein. For rat TGF-β1, Western blot analysis compared recombinant protein against commercial standard to verify antigenicity .

• Activity verification through purification steps: Track specific activity through purification to ensure functionality is maintained, as demonstrated with rat monoamine oxidase B:

How can I improve solubility when recombinant rat CutA forms inclusion bodies?

Inclusion body formation is common with recombinant protein expression in bacterial systems:

• Expression conditions: Lower temperature (16-20°C), reduced inducer concentration, and slower induction can improve folding and solubility.

• Solubility tags: Fusion to MBP, SUMO, or TrxA can enhance solubility significantly.

• Co-expression with chaperones: Co-express with folding chaperones like GroEL/ES, DnaK/J, or trigger factor.

• Inclusion body refolding: If inclusion bodies persist, develop a refolding protocol. For rat TGF-β1, inclusion bodies were solubilized in 6M guanidine hydrochloride with reducing agent, followed by gel filtration chromatography to achieve homogeneity .

• Switch expression systems: Consider yeast or mammalian expression systems that often provide better folding environments for mammalian proteins .

What strategies help resolve expression heterogeneity in recombinant rat proteins?

Heterogeneity in recombinant protein expression can complicate structural and functional studies:

• Construct optimization: Systematic variation of N- and C-termini to identify stable domains. For rat NK cell receptors, multiple constructs with varying terminus positions resulted in different expression patterns and oligomeric states .

• Post-translational processing: Incomplete processing can cause heterogeneity. Recombinant rat protein C displayed 70-90% two-chain form (41 kDa heavy chain; 22 and 23 kDa light chain) and 10-30% single-chain (61 kDa), while plasma rat PC was exclusively in two-chain form .

• Glycosylation control: Uniform glycosylation can be achieved through expression in glycosylation-deficient cell lines or enzymatic deglycosylation post-purification.

• Proteolytic stability: Add protease inhibitors during purification. For studying rat protein C secretion, aprotinin was included in the culture medium to prevent proteolysis .

• Chromatographic separation: Employ multiple orthogonal chromatography steps to separate different forms. For rat protein C, heterogeneity in the light chain (22 and 23 kDa forms) was observed in recombinant but not plasma-derived protein .

• Mass spectrometry: Identify the molecular basis of heterogeneity through mass spectrometric analysis of different protein forms.

How do species-specific differences affect the application of rat CutA findings to human systems?

Cross-species applicability requires careful consideration:

• Sequence homology analysis: Compare rat and human CutA sequences to identify conserved and divergent regions, which may affect functional conservation.

• Functional conservation testing: Directly compare rat and human proteins in the same assay. For protein C, both recombinant and plasma rat PC prolonged clotting time in rat plasma, while human PC was less effective, highlighting species-specific functional differences .

• Structural comparison: If structures are available, compare rat and human proteins to identify structural differences that may affect function or ligand binding.

• Expression pattern differences: Analyze tissue-specific expression patterns across species, as these may indicate different physiological roles.

• Interaction partner conservation: Determine whether interaction partners are conserved between species, as differences may limit translational relevance.

• Post-translational modification differences: Compare PTM patterns between species, as these can significantly affect function.

What are the latest methodological advances in recombinant rat protein structural biology?

Recent technological advances have expanded our capabilities:

• Cryo-electron microscopy (cryo-EM): Recent advances have revolutionized structural studies of challenging proteins, allowing near-atomic resolution without crystallization.

• Integrative structural biology: Combining multiple techniques (X-ray crystallography, NMR, cryo-EM, SAXS) provides more comprehensive structural insights.

• Serial crystallography: X-ray free-electron lasers (XFELs) enable structure determination from microcrystals or time-resolved studies.

• Membrane mimetics: Nanodiscs, lipidic cubic phase, and styrene-maleic acid copolymer lipid particles (SMALPs) provide better environments for membrane protein structural studies .

• Fluorescence-detected size exclusion chromatography (FSEC): This technique has significantly advanced membrane protein research by allowing rapid screening of construct stability and monodispersity .

• Computational advances: AlphaFold and other AI-based structure prediction methods have dramatically improved our ability to model protein structures.

• Gene editing: CRISPR/Cas9 enables precise engineering of expression hosts for improved protein production.

The rapid evolution of these technologies continues to expand our capabilities for studying challenging proteins like CutA.