Recombinant Cat Sex-determining region Y protein (SRY)

What is the structural and functional characterization of feline SRY protein?

Feline SRY is a single-copy gene located on the Y chromosome that triggers male sex determination during embryonic development. The gene consists of approximately 705 bp in the cat family and encodes a DNA-binding high mobility group (HMG) box transcription factor . The cat SRY protein contains regions similar to those found in other mammals:

• HMG-box domain: Critical for DNA binding

• Bridge region: Important for protein-protein interactions

• N-terminal and C-terminal regions: May have regulatory functions

Purification strategies should be tailored to both the expression system and the downstream applications:

• Affinity chromatography: Most common first-step purification using:

  • His-tagged SRY: Ni-NTA or IMAC columns

  • GST-tagged SRY: Glutathione Sepharose columns

• Buffer optimization for solubility:

  • Tris-based buffers (pH 7.4-8.0) containing:

    • 50 mM KCl

    • 10 mM reduced glutathione (for GST-tagged proteins)

    • 50% glycerol for storage stability

• Quality assessment methods:

  • SDS-PAGE with Coomassie Blue or silver staining (target purity >90%)

  • Western blotting for identity confirmation

  • Activity assays to confirm DNA-binding functionality

Researchers should consider incorporating solubility enhancers during purification, especially when working with full-length SRY, which can form inclusion bodies in bacterial systems .

How can researchers optimize expression conditions to improve solubility of recombinant cat SRY protein?

Studies on recombinant bovine SRY reveal strategies that may be applicable to feline SRY :

Induction parameters optimization:

• Temperature: Lower temperatures (27-32°C) significantly increase soluble protein yield compared to standard 37°C induction

• IPTG concentration: Lower concentrations (0.3 mM) produce more soluble protein compared to high concentrations (1.2 mM)

Genetic optimization approaches:

• Codon optimization: Significantly improves soluble protein yield in E. coli expression systems by adapting codon usage to the host organism

• Expression of partial constructs: Using only the HMG-box domain (positions 1-144 aa in mouse SRY) can improve solubility while maintaining DNA-binding activity

Media composition modifications:

• Adding stabilizers to cultivation media

• Using enriched media formulations

• Incorporating solubility enhancers during protein expression

A comparative analysis found that cultivating codon-optimized SRY at 32°C with 0.3 mM IPTG produced significantly more soluble protein than wild-type constructs, with substantially reduced inclusion body formation .

What molecular interactions mediate SRY's role in sex determination pathways, and how can they be studied using recombinant protein?

SRY participates in several key molecular interactions that can be investigated using recombinant protein:

SRY-DNA interactions:

• SRY binds to the core sequence AACAAAG in a sequence-dependent manner, similar to the T-cell specific protein TCF-1

• Recombinant SRY can be used in electrophoretic mobility shift assays (EMSAs) to study sequence specificity and binding affinities

• DNA-binding activity of SRY is essential for sex determination, as demonstrated by mutations in XY females that reduce or eliminate DNA binding

SRY-β-catenin interactions:

• SRY inhibits β-catenin-mediated Wnt signaling through direct protein-protein interaction

• This interaction is independent of SRY's DNA-binding and transactivation functions

• SRY and β-catenin colocalize in specific nuclear bodies, suggesting a mechanism where SRY sequesters β-catenin

SRY-KRAB domain protein interactions:

• SRY interacts with KRAB-O (KRAB Only), a protein containing only a Krüppel-associated box domain

• This interaction maps to the bridge region outside the HMG box

• Through KRAB-O, SRY associates with KAP1 (KRAB-associated protein 1) and heterochromatin protein 1 (HP1)

These interactions suggest that SRY regulates gene expression through both direct DNA binding and by modulating other transcriptional regulatory complexes, which may be critical for its role in sex determination.

How does recombinant SRY protein expression differ between wild-type and mutant forms associated with sex reversal?

Studies of SRY mutations in XY females provide insights into structure-function relationships:

DNA-binding mutations:

• Four SRY mutant proteins from XY females with defective DNA binding activity showed negligible binding to target sequences in vitro

• Surprisingly, these mutants retained near wild-type inhibitory activity against β-catenin, suggesting DNA binding is not required for all SRY functions

Nuclear localization mutations:

• Three SRY mutant proteins with nuclear localization defects failed to inhibit β-catenin, indicating proper subcellular localization is critical

• These mutants provide tools for studying non-transcriptional functions of SRY

Transactivation domain mutations:

• SRY-VP16 fusion protein (containing a potent transactivator domain) showed wild-type inhibitory activity against β-catenin, suggesting transactivation is not required for this function

When working with recombinant SRY protein, researchers should consider:

• Including wild-type controls alongside mutant forms

• Assessing both DNA-binding and protein-interaction capabilities

• Evaluating proper folding and nuclear localization signals

• Testing multiple functional readouts, as mutations may affect some functions while preserving others

What evolutionary insights can be gained from comparative analysis of recombinant SRY proteins across the cat family?

Phylogenetic analyses of SRY across 36 species in the cat family Felidae provide several key insights :

Structural variations:

• Four different species have significantly altered SRY proteins due to insertion/deletion events:

  • Ocelot (L. pardalis): A single-base-pair insertion at position 64 resulting in a frameshift, potentially using an alternate ATG start codon to produce a shortened protein (by 7 residues)

  • Clouded leopard (N. nebulosa): A single-base-pair deletion at position 437 resulting in a truncated protein

Methodological approach for evolutionary studies:

• Amplify SRY and flanking regions using PCR with Y-chromosome-specific primers

• Sequence the entire coding region and adjacent genomic flanking regions

• Analyze selection pressures using models that calculate nonsynonymous/synonymous substitution ratios

• Implement phylogenetic reconstruction using multiple optimality criteria (ME, ML, MP)

• Test for variable selection pressure among sites using different statistical models (M0, M1a, M2a, M7, M8)

These approaches allow researchers to understand how SRY has evolved within the cat family and identify specific regions under selection that may contribute to species-specific aspects of sex determination.

What methods are most effective for analyzing the functional activity of recombinant cat SRY protein?

Multiple complementary approaches can assess the functionality of recombinant SRY:

DNA-binding assays:

• Electrophoretic Mobility Shift Assays (EMSAs) to determine binding to the core sequence AACAAAG recognized by SRY

• Chromatin Immunoprecipitation (ChIP) to identify genomic binding sites in cells

• Surface Plasmon Resonance (SPR) to measure binding kinetics and affinities

Transcriptional regulation assays:

• Reporter gene assays using TCF-dependent promoters to measure SRY inhibition of β-catenin-mediated transcription

• Quantitative RT-PCR to measure expression of putative SRY target genes

• RNA-seq analysis following SRY introduction into relevant cell types

Protein-protein interaction assays:

• Co-immunoprecipitation to confirm interactions with partners like β-catenin and KRAB-O

• GST pull-down assays for direct binding studies

• Yeast two-hybrid screens to identify novel interacting proteins

• Immunofluorescence colocalization studies to visualize SRY with interaction partners in nuclear bodies

Functional cell-based assays:

• Nuclear translocation assays to assess localization sequences

• β-catenin localization studies to observe SRY-triggered relocalization into specific nuclear bodies

Results from the HEK293T cell model show that wild-type SRY inhibits β-catenin-mediated TCF-dependent gene activation in the presence of GSK3β inhibitors or activated β-catenin mutants, providing a valuable functional readout .

How can recombinant SRY protein be used to develop sex-determination assays in feline conservation and research?

Recombinant SRY protein can contribute to feline conservation and research in several ways:

Development of SRY antibodies:

• Purified recombinant SRY can be used to develop specific antibodies for:

  • Immunohistochemistry of gonadal tissues

  • Western blot analysis of tissue samples

  • ELISA-based sex determination from minimally invasive samples

Molecular diagnostic tools:

• Knowledge of SRY sequence variation across felid species can inform PCR primer design for:

  • Species-specific sex determination assays

  • Detection of SRY mutations associated with disorders of sex development

  • Non-invasive genetic sampling for wildlife conservation

Functional validation studies:

• Recombinant wild-type and mutant SRY proteins can be used to:

  • Test effects of naturally occurring SRY variations in cats

  • Validate the pathogenicity of novel SRY mutations

  • Develop in vitro models for feline gonadal development

Methods for protein design and production:

• First, determine the full-length sequence of cat SRY from genomic DNA

• Design constructs with appropriate tags (His, GST) for easy purification

• Consider codon optimization to enhance expression in E. coli

• Express at lower temperatures (27-32°C) with moderate IPTG (0.3mM) to improve solubility

• Purify using affinity chromatography followed by size exclusion

• Validate functionality through DNA binding and protein interaction assays